Nicole Willems

Inflammatory profiles in canine intervertebral disc degeneration

BackgroundIntervertebral disc (IVD) disease is a common spinal disorder in dogs and degeneration and inflammation are significant components of the pathological cascade. Only limited studies have studied the cytokine and chemokine profiles in IVD degeneration in dogs, and mainly focused on gene expression. A better understanding is needed in order to develop biological therapies that address both pain and degeneration in IVD disease. Therefore, in this study, we determined the levels of prostaglandin E2 (PGE2), cytokines, chemokines, and matrix components in IVDs from chondrodystrophic (CD) and non-chondrodystrophic (NCD) dogs with and without clinical signs of IVD disease, and correlated these to degeneration grade (according to Pfirrmann), or herniation type (according to Hansen). In addition, we investigated cyclooxygenase 2 (COX-2) expression and signs of inflammation in histological IVD samples of CD and NCD dogs.ResultsPGE2 levels were significantly higher in the nucleus pulposus (NP) of degenerated IVDs compared with non-degenerated IVDs, and in herniated IVDs from NCD dogs compared with non-herniated IVDs of NCD dogs. COX-2 expression in the NP and annulus fibrosus (AF), and proliferation of fibroblasts and numbers of macrophages in the AF significantly increased with increased degeneration grade. GAG content did not significantly change with degeneration grade or herniation type. Cytokines interleukin (IL)-2, IL-6, IL-7, IL-8, IL-10, IL-15, IL-18, immune protein (IP)-10, tumor necrosis factor (TNF)-α, and granulocyte macrophage colony-stimulating factor (GM-CSF) were not detectable in the samples. Chemokine (C-C) motif ligand (CCL)2 levels in the NP from extruded samples were significantly higher compared with the AF of these samples and the NP from protrusion samples.ConclusionsPGE2 levels and CCL2 levels in degenerated and herniated IVDs were significantly higher compared with non-degenerated and non-herniated IVDs. COX-2 expression in the NP and AF and reactive changes in the AF increased with advancing degeneration stages. Although macrophages invaded the AF as degeneration progressed, the production of inflammatory mediators seemed most pronounced in degenerated NP tissue. Future studies are needed to investigate if inhibition of PGE2 levels in degenerated IVDs provides effective analgesia and exerts a protective role in the process of IVD degeneration and the development of IVD disease.

Biocompatibility and intradiscal application of a thermoreversible celecoxib-loaded poly-N-isopropylacrylamide MgFe-layered double hydroxide hydrogel in a canine model.

IntroductionChronic low back pain due to intervertebral disc (IVD) degeneration is associated with increased levels of inflammatory mediators. Current medical treatment consists of oral anti-inflammatory drugs to alleviate pain. In this study, the efficacy and safety of a novel thermoreversible poly-N-isopropylacrylamide MgFe-layered double hydroxide (pNIPAAM MgFe-LDH) hydrogel was evaluated for intradiscal controlled delivery of the selective cyclooxygenase (COX) 2 inhibitor and anti-inflammatory drug celecoxib (CXB).MethodsDegradation, release behavior, and the ability of a CXB-loaded pNIPAAM MgFe-LDH hydrogel to suppress prostaglandin E2 (PGE2) levels in a controlled manner in the presence of a proinflammatory stimulus (TNF-α) were evaluated in vitro. Biocompatibility was evaluated histologically after subcutaneous injection in mice. Safety of intradiscal application of the loaded and unloaded hydrogels was studied in a canine model of spontaneous mild IVD degeneration by histological, biomolecular, and biochemical evaluation. After the hydrogel was shown to be biocompatible and safe, an in vivo dose–response study was performed in order to determine safety and efficacy of the pNIPAAM MgFe-LDH hydrogel for intradiscal controlled delivery of CXB.ResultsCXB release correlated to hydrogel degradation in vitro. Furthermore, controlled release from CXB-loaded hydrogels was demonstrated to suppress PGE2 levels in the presence of TNF-α. The hydrogel was shown to exhibit a good biocompatibility upon subcutaneous injection in mice. Upon intradiscal injection in a canine model, the hydrogel exhibited excellent biocompatibility based on histological evaluation of the treated IVDs. Gene expression and biochemical analyses supported the finding that no substantial negative effects of the hydrogel were observed. Safety of application was further confirmed by the absence of clinical symptoms, IVD herniation or progression of degeneration. Controlled release of CXB resulted in a nonsignificant maximal inhibition (approximately 35 %) of PGE2 levels in the mildly degenerated canine IVDs.ConclusionsIn conclusion, this study showed biocompatibility and safe intradiscal application of an MgFe LDH-pNIPAAM hydrogel. Controlled release of CXB resulted in only limited inhibition of PGE2 in this model with mild IVD degeneration, and further studies should concentrate on application of controlled release from this type of hydrogel in animal models with more severe IVD degeneration.

Detection and Quantification of β-Amyloid, Pyroglutamyl Aβ, and Tau in Aged Canines

Abstract Canine cognitive dysfunction syndrome is an age-associated disorder that resembles many aspects of human Alzheimer disease. The characterization of canine cognitive dysfunction syndrome has been restricted to selected laboratory dogs and mongrels, thereby limiting our knowledge of potential breed-related and age-related differences. We examined the brains of 24 dogs from various breeds. The frontal cortex, hippocampus, and entorhinal cortex were investigated. Deposits of &bgr;-amyloid (A&bgr;) and tau were analyzed phenotypically and quantified stereologically. In all dogs aged 10 years or older, plaques containing pyroglutamyl A&bgr; and A&bgr;8–17 were detected. Within the ventral hippocampus, significantly more pyroglutamyl A&bgr; plaques were deposited in small and medium dogs than in large dogs. Hyperphosphorylated tau with formation of neurofibrillary tangles was observed in 3 animals aged 13 to 15 years. This study provides the first investigation of pyroglutamyl A&bgr; in comparison with total A&bgr; (as shown by A&bgr;8–17 immunoreactivity) in dogs of different breeds, sizes, and ages. Our results indicate that canine cognitive dysfunction syndrome is relatively common among aged canines, thereby emphasizing the relevance of such populations to translational Alzheimer disease research.

A novel injectable thermoresponsive and cytocompatible gel of poly(N-isopropylacrylamide) with layered double hydroxides facilitates siRNA delivery into chondrocytes in 3D culture

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.

Intradiscal application of a PCLA–PEG–PCLA hydrogel loaded with celecoxib for the treatment of back pain in canines : What's in it for humans?

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.

Safety of intradiscal injection and biocompatibility of polyester amide microspheres in a canine model predisposed to intervertebral disc degeneration.

Abstract Repair of degenerated intervertebral discs (IVD) might be established via intradiscal delivery of biologic therapies. Polyester amide polymers (PEA) were evaluated for in vitro cytotoxicity and in vivo biocompatibility, and thereafter intradiscal application of PEA microspheres (PEAMs) in a canine model predisposed to IVD degeneration at long‐term (6 months) follow‐up. PEA extracts did not induce cytotoxicity in mouse fibroblast cells (microscopy and XTT assay), while a slight foreign body reaction was demonstrated by histopathology after intramuscular implantation in rabbits. Intradiscal injection of a volume of 40 µL through 26 and 27G needles induced no degenerative changes in acanine model susceptible to IVD disease. Although sham‐injected IVDs showed increased CAV1 expression compared with noninjected IVDs, which may indicate increased cell senescence, these findings were not supported by immunohistochemistry, biomolecular analysis of genes related to apoptosis, biochemical and histopathological results. PEAM‐injected IVDs showed significantly higher BAX/BCL2 ratio vs sham‐injected IVDs suggestive of an anti‐apoptotic effect of the PEAMs. These findings were not supported by other analyses (clinical signs, disc height index, T2 values, biomolecular and biochemical analyses, and IVD histopathology). PEAs showed a good cytocompatibility and biocompatibility. PEAMs are considered safe sustained release systems for intradiscal delivery of biological treatments.

Disseminated Candidiasis in a Young, Previously Healthy, Dog and Review of Literature.

BackgroundThe reports on disseminated candidiasis in dogs so far describe at least one predisposing factor. This case report, however, highlights candidiasis in a dog without any known predisposition.PatientA 1.5-year-old intact female Hovawart dog was presented with subcutaneous nodules and polyuria/polydipsia. An excisional biopsy revealed a chronic pyogranulomatous and necrotizing inflammation with mycotic structures. The patient became febrile and lethargic, and developed lameness.MethodsA physical examination, blood tests, urinalysis, thoracic radiographs, abdominal ultrasonography of the abdomen, fine-needle aspiration biopsies, and a culture of a subcutaneous nodule aspirate were obtained. Selected sections of multiple organs were collected for routine histology postmortem. The isolate and a subcutaneous mass were subjected to molecular identification and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF–MS) analysis.ResultsClinical, laboratory, and radiological findings were consistent with a granulomatous chronic systemic inflammation. Cytology and histology showed a pyogranulomatous and necrotizing inflammation with myriads of intra- and extra-cellular yeasts and extracellular hyphae. Culture yielded numerous yeast colonies, which appeared Candida albicans–like, but showed a negative serum test and a low identification in API 20 C AUX. Nucleic acid sequences showed homology with the C. albicans-type strain CBS 562. Multilocus sequence typing (MLST) resulted in a new type with designation DST121. The identification of the isolates was confirmed by MALDI-TOF–MS analysis.Conclusion and Clinical ImportanceFuture MLST typing and investigation of virulence can provide further evidence whether this MLST-type is associated with clinical cases of disseminated candidiasis without an apparent predisposing condition.

Temporary Segmental Distraction in a Dog with Degenerative Lumbosacral Stenosis

OBJECTIVES  Degenerative lumbosacral stenosis (DLSS) is characterized by intervertebral disc degeneration and causes lower back pain in dogs. Temporary distraction in rabbit models with induced intervertebral disc degeneration showed signs of intervertebral disc repair. In the present study, we assessed safety and efficacy of temporary segmental distraction in a dog with clinical signs of DLSS. METHODS  Distraction of the lumbosacral junction by pedicle screw-rod fixation was applied in a 5-year-old Greyhound with DLSS and evaluated by radiography, magnetic resonance imaging, and force plate analysis before and after distraction. RESULTS  Safe distraction of the lumbosacral junction was demonstrated, with improvement of clinical signs after removal of the distraction device. Signal intensity of the intervertebral disc showed no changes over time. T2 value was highest directly after removal of the distraction device but decreased by 10% of the preoperative value at 9 months of follow-up. Disc height decreased (8%) immediately after removal of the distraction device, but recovered to the initial value. A decrease in the pelvic/thoracic propulsive force during pedicle screw-rod fixation and distraction was demonstrated, which slowly increased by 4% compared with the initial value. CLINICAL SIGNIFICANCE  Temporary pedicle screw-rod fixation in combination with distraction in a dog with DLSS was safe, improved clinical signs and retained disc height at 9 months of follow-up.

Intradiscal Injection of a Slow Release Formulation of Celecoxib for the Treatment of Dogs with Low Back Pain

Introduction Intervertebral disc (IVD) disease characterized by low back pain is common in both humans and large breed dogs. Inflammatory mediators such as prostaglandin E2 (PGE2) play a key role in IVD degeneration, causing structural changes of the IVD and low back pain. The current conservative and surgical treatment modalities do not reverse IVD degeneration and have certain drawbacks. Long term systemic administration of non-steroidal anti-inflammatory drugs can cause gastro-intestinal side effects. Decompressive surgery is effective in over 80% of the patients, but is rather costly and has a long recovery period. Injectable formulations that enable local sustained release of anti-inflammatory drugs aim at decreasing inflammation and thereby inhibiting degeneration and pain. In vitro, controlled release of celecoxib, a COX-2 inhibitor, from a thermoreversible acetyl-capped PCLA-PEG-PCLA hydrogel inhibited PGE2 production and enhanced matrix production for 28 days in 3D culture of canine nucleus pulposus cells that were subjected to the catabolic effects of TNF-α. In vivo, sustained release of celecoxib by the hydrogel for up to 60 days was shown and safely applied intradiscally in laboratory dogs with naturally occurring IVD degeneration. The aim of the present study is to report the safety and feasibility of intradiscal application in client-owned dogs with low back pain related to IVD degeneration. Material and Methods Client-owned dogs with low back pain were diagnosed by MRI with degenerative lumbosacral stenosis marked by mild to moderate IVD degeneration and protrusion. The dogs were surgical candidates but were offered intradiscal injection. The PCLA-PEG-PCLA thermogel, loaded with 0.013mg/ml celecoxib, was percutaneously injected into the nucleus pulposus of L7-S1 under fluoroscopy guidance. Follow-up consisted of clinical examination, owner questionnaires, and objective gait analysis by measurement of ground reaction forces (GRFs) using force plate analysis (FPA) at 6 weeks, and 3 and 6 months after injection. MRI was repeated after 3 months and included T2- and T1-weighted images and T2-mapping. Results Ten dogs with DLLS were injected with the celecoxib-loaded hydrogel. None of the dogs showed adverse reactions after intradiscal injection. Follow up MRI showed no worsening of the IVD degeneration. Clinical improvement was achieved by reduction of low back pain in 9/10 dogs, as was shown by clinical examination, force plate analysis and owner questionnaires. In 3/10 dogs low back pain recurred at 3 months and they subsequently underwent standard-of-care surgical treatment. Conclusion This study showed the safety and feasibility of intradiscal injections with a thermoresponsive hydrogel loaded with celecoxib. Ongoing studies concentrate on the long term clinical follow up of these patients. Future studies will determine the optimal loading dose of celecoxib for clinical efficacy. In this setup, 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.

Pedicle Screw-Rod Fixation for Treatment of Lumbosacral Intervertebral Disc Degeneration in Dogs with Low Back Pain

Introduction Low back pain in nonchondrodystrophic large breed dogs is a common problem and the most common cause is degenerative lumbosacral stenosis (DLSS). DLSS is characterized by intervertebral disc (IVD) degeneration of L7-S1, disc bulging, disc herniation, spinal canal stenosis, compression of neuronal tissue (cauda equina), thickened nerve roots (neuritis), soft tissue and bony proliferations, and foraminal stenosis. This ultimately results in discogenic and neurogenic pain. DLSS in dogs maybe treated conservatively or surgically. The most common surgical treatment is decompressive dorsal laminectomy and partial discectomy but in severe degenerated IVDs or in cases with foraminal stenosis and neuritis this may not be sufficient to treat low back pain. In these cases, pedicle screw-rod fixation (PSRF) can be performed. The aim of PSRF was to increase the intervertebral foraminal space for the exiting nerves and to promote spinal fusion. Distraction of the peripheral joints is one of the most promising new techniques in allowing chondrocytes to initiate repair of damaged cartilage in osteoarthritis. IVD degeneration, such as osteoarthritis, is also characterized by a decreasing number of nucleus pulposus cells and disc matrix. PSRF allows distraction of the IVD and may initiate, in a similar fashion as in joints, repair of the disc matrix. This was investigated in a pilot study in a dog that underwent temporary distraction and follow-up with magnetic resonance imaging (MRI). The aim of the present study was to investigate the effectiveness of PSRF in dogs with severe lumbosacral disc degeneration, to assess the effect of PSRF on the intervertebral foramina and on spinal fusion and, in a pilot study, to investigate the regenerative role of temporary distraction on the degenerated lumbosacral disc. Materials and Methods Eleven dogs with confirmed diagnosis of DLSS and IVD degeneration underwent PSRF of the lumbosacral junction (Fig. 1). In 10 (out of the 11) dogs, PSRF was combined with dorsal laminectomy, disc fenestration, and various techniques to promote spinal fusion. In a subset of these patients (4 of 10 dogs), permanent distraction of the lumbosacral joint before PSRF was applied. In order to investigate the effect of temporary distraction of the degenerated lumbosacral joint, the PSRF was applied on the distracted joint for 3 months and removed thereafter. During follow-up, the dogs were monitored using questionnaires to owners and by clinical evaluation, diagnostic imaging with computed tomography and MRI, and measurement of ground reaction forces by force plate analysis. In the 4 dogs in which permanent distraction was applied, the increase in the intervertebral foramen was measured and in 1 dog the effect of temporary distraction on the disc matrix was evaluated on MRI. Results Clinical evaluation, responses of questionnaires to owners, and force plate data showed improvement or complete resolution of clinical signs after pedicle screw-rod fixation in 11 dogs during follow-up periods ranging from 5 months to 4 years. Diagnostic imaging showed no implant failures. In none of the 10 dogs was intervertebral body bone fusion of the lumbosacral joint achieved. In the dogs in which permanent distraction was applied, postoperative imaging showed significant enlargement of the intervertebral foramen between L7 and S1 (Fig. 1). In the dog with temporary distraction, MRI indicated no changes in the IVD signal 3 months after removal of the PSRF device. Fig. 1 Canine lumbosacral spine segment in a dog with degenerative lumbosacral stenosis before and after distraction plus pedicle screw-rod fixation (PSRF). Conclusion Pedicle screw-rod fixation offers a surgical treatment option for large dogs with severe degenerative lumbosacral stenosis. However, PSRF alone does not result in intervertebral body fusion between L7 and S1 and for that additional methods are necessary. Distraction and PSRF is effective in enlarging the intervertebral foramina and alleviating clinical signs. Temporary distraction alone, to initiate regeneration of the intervertebral disc matrix, did not have adverse effects and is an interesting concept for further investigation. Disclosure of Interest None declared