Canine degenerative myelopathy : perspectives from aging, microglia and neurofilaments

[ACCESS RESTRICTED TO THE UNIVERSITY OF MISSOURI AT REQUEST OF AUTHOR.] Canine degenerative myelopathy (DM) is a late-onset neurodegenerative disease similar to some forms of amyotrophic lateral sclerosis (ALS). Mutations in superoxide dismutase 1 (SOD1) are a risk factor for disease; however, disease penetrance is also age-related. Microglia, the immune effector cells of the CNS, undergo phenotypic changes with age that may predispose to neurodegeneration. Furthermore, they have been directly implicated in disease pathogenesis in ALS. We tested the hypotheses that 1) microglia in aged, neurologically normal dogs have increased activation toward a neurotoxic phenotype compared to adult dogs and 2) microglia in DM-affected dogs will transition from a neuroprotective (M2) to neurotoxic (M1) phenotype as disease progresses and 3) increased fractalkine, a chemotactic molecule for microglia, will correlate with increased M2 microglia in DM-affected dogs. We found that aged, neurologically normal dogs had increased numbers of M1 and M2-activated microglia; however, total M2 microglia per motor neuron was reduced from adult dogs. Furthermore, compared to adult dogs, aged canine spinal cord had increased expression of genes associated with phagocytosis and proteolysis, which also are elevated in ALS and Alzheimer's disease models. Within the context of DM, microglia in close proximity to motor neurons were increased at all disease stages. M2 microglia per motor neuron were increased in early stages of DM, whereas the number of M1 microglia per motor neuron were indistinguishable from aged controls at all stages of disease. Fractalkine levels did not change throughout disease progression. These findings collectively suggested that aged canine microglia are highly activated with reduced neuroprotective microglia near motor neurons. In the context of DM, concomitant loss of motor function and increased M2 microglia, without a change in M1 microglia, suggested that SOD1E40K M2 microglia could have contributed to neurodegeneration through a toxic gain of function. Currently, there is no definitive ante-mortem diagnostic test for DM. We tested the hypothesis that phosphorylated neurofilament heavy (pNF-H), an abundant structural protein of myelinated motor axons, is readily detectable in CSF and serum of DM-affected dogs, and can be used as a diagnostic biomarker for DM. We identified pNF-H as a sensitive and specific diagnostic test for SOD1E40K-associated canine DM. This work may provide the first ante-mortem diagnostic test for canine DM.Includes biblographical reference

Prevalence and Clinicopathologic Features of Canine Metastatic Melanoma Involving the Central Nervous System: A Retrospective Analysis and Comparative Review.

BackgroundCentral nervous system (CNS) involvement is the leading cause of death in malignant melanoma. Rodent models, while vital to mechanistic investigation, have had limited success identifying effective therapies for melanoma brain metastases. The companion dog with de novo melanoma is a promising complementary model for developmental therapeutic investigation, as these tumors occur in an immunologically outbred host that has shared environmental exposures with humans. However, relatively little is known regarding the prevalence and clinicopathological features of canine melanoma metastasis to the CNS. To further validate the dog as an appropriate model for human metastatic melanoma, the aims of this study were to determine the rate of CNS metastasis and associated clinicopathologic features in canine malignant melanoma.MethodsMedical records of dogs diagnosed with malignant melanoma from 1985-2019 at the University of California Davis Veterinary Medical Teaching Hospital were assessed retrospectively. Clinicopathologic features were compared between dogs with CNS metastasis (CNS+) and dogs without CNS metastasis (CNS-). Site of CNS involvement and associated neurological signs were analyzed via Wilcoxon-Mann-Whitney rank sum and Fisher's exact tests. Survival data were analyzed via Kaplan-Meier estimates.ResultsCNS metastasis was identified in 38% of dogs in this study (20/53). The oral cavity was the most common site of primary melanoma in both groups [CNS+: n=12 (60%) vs. CNS-: n=22 (67%); p>0.99]. The total burden of metastatic disease was higher in the CNS+ group (CNS+: 4, 95% CI 3-5 vs. CNS-: 3, 95% CI 1-3; p<0.001). The cerebrum was the most common site of CNS metastasis (n=15, 75%) and seizures were the most observed neurological sign (n=9, 64%). There was no difference in overall survival between CNS+ and CNS- groups. However, the median survival time following onset of neurological signs was 9.5 days (95% CI 1-43), with 5 dogs euthanized within 24 hours of the onset of neurological signs.ConclusionsCanine and human MM patients share similar rates of CNS metastasis and clinical presentation. This study will guide clinical management of canines with malignant melanoma and inform future studies using dogs with spontaneously occurring melanoma as a preclinical model for human melanoma brain metastases

Microglia-Derived Olfactomedin-like 3 Promotes Pro-Tumorigenic Microglial Function and Malignant Features of Glioma Cells.

Under the influence of transforming growth factor-beta (TGFβ), glioma-associated microglia produce molecules that promote glioma growth and invasion. Olfactomedin-like 3 (Olfml3), a novel, secreted glycoprotein, is known to promote several non-CNS cancers. While it is a direct TGFβ1 target gene in microglia, the role of microglia-derived OLFML3 in glioma progression is unknown. Here, we tested the hypotheses that microglial Olfml3 is integral to the pro-tumorigenic glioma-associated microglia phenotype and promotes glioma cell malignancy. Using an Olfml3 knockout microglial cell line (N9), we demonstrated that Olfml3 is a direct target gene of all TGFβ isoforms in murine microglia. Moreover, loss of Olfml3 attenuated TGFβ-induced restraint on microglial immune function and production of cytokines that are critical in promoting glioma cell malignancy. Importantly, microglia-derived OLFML3 directly contributes to glioma cell malignancy through increased migration and invasion. While exposure to conditioned medium (CM) from isogenic control microglia pre-treated with TGFβ increased mouse glioma cell (GL261) migration and invasion, this effect was abolished with exposure to CM from TGFβ-treated Olfml3-/- microglia. Taken together, our data suggest that Olfml3 may serve as a gatekeeper for TGFβ-induced microglial gene expression, thereby promoting the pro-tumorigenic microglia phenotype and glioma cell malignancy

Idiopathic functional urinary outflow tract obstruction in dogs, a retrospective case series (2010-2021): 31 cases.

BACKGROUND: Idiopathic functional urinary outflow tract obstruction (iFUOTO) is an uncommon but life-limiting disease whose etiology and clinical course of disease remain poorly understood. OBJECTIVE: Characterize signalment, clinical signs, clinicopathologic findings, treatments, and propose a standardized response score for dogs with iFUOTO. ANIMALS: Thirty-one client-owned dogs diagnosed with iFUOTO. METHODS: Retrospective case series. Medical records from 2010 to 2021 were reviewed and findings recorded. Dogs were categorized based on the presence or absence of overt signs of neurological disease. Response to treatment was scored. RESULTS: Thirty-one dogs were included. All dogs had stranguria and 14 (45%) had overflow urinary incontinence. Mean age of onset for signs was 6.9 years±8 months. Twenty-four dogs (77%) were castrated males, 5 (16%) intact males, and 2 (6%) spayed females. Eight (26%) of dogs had overt neurological deficits. Follow-up data were available for 29 dogs (median 38 days, range: 2-1277). Final outcome scores were not significantly different among dogs with overt signs of neurological disease (median score 2.5; range: 0-3) and those without (median score 1; range; 0-3; P = .35). Treatments included alpha antagonists, skeletal muscle relaxants, parasympathomimetics, anti-inflammatories, castration, temporary placement of a urethral catheter, or a combination of these. CONCLUSIONS AND CLINICAL RELEVANCE: Multimodal treatment was frequently prescribed, but a standard outcome score is needed to evaluate the effectiveness of these therapies. Serial neurological examinations and monitoring of the dogs dysuria are warranted

Microglia-Derived Olfactomedin-like 3 Is a Potent Angiogenic Factor in Primary Mouse Brain Endothelial Cells: A Novel Target for Glioblastoma.

Neoangiogenesis, a hallmark feature of all malignancies, is robust in glioblastoma (GBM). Vascular endothelial growth factor (VEGF) has long been regarded as the primary pro-angiogenic molecule in GBM. However, anti-VEGF therapies have had little clinical efficacy, highlighting the need to explore VEGF-independent mechanisms of neoangiogenesis. Olfactomedin-like 3 (OLFML3), a secreted glycoprotein, is an established proangiogenic factor in many cancers, but its role in GBM neoangiogenesis is unknown. To gain insight into the role of OLFML3 in microglia-mediated angiogenesis, we assessed endothelial cell (EC) viability, migration and differentiation following (1) siRNA knockdown targeting endogenous EC Olfml3 and (2) EC exposure to human recombinant OLFML3 (rhOLFML3; 10 ng/mL, 48 h), and conditioned medium (CM) from isogenic control and Olfml3-/- microglia (48 h). Despite a 70% reduction in Olfml3 mRNA levels, EC angiogenic parameters were not affected. However, exposure to both rhOLFML3 and isogenic control microglial CM increased EC viability (p < 0.01), migration (p < 0.05) and differentiation (p < 0.05). Strikingly, these increases were abolished, or markedly attenuated, following exposure to Olfml3-/- microglial CM despite corresponding increased microglial secretion of VEGF-A (p < 0.0001). Consistent with reports in non-CNS malignancies, we have demonstrated that OLFML3, specifically microglia-derived OLFML3, promotes VEGF-independent angiogenesis in primary brain microvascular ECs and may provide a complementary target to mitigate neovascularization in GBM

Serum phosphorylated neurofilament heavy chain as a diagnostic biomarker for progressive myelomalacia in dogs with thoracolumbar intervertebral disc herniation.

BackgroundSerum phosphorylated neurofilament-heavy chain (pNF-H) has not been longitudinally evaluated in dogs that develop progressive myelomalacia (PMM) after Type I intervertebral disc herniation (IVDH).ObjectivesTo determine if serum pNF-H concentrations would predict outcome of neuroligical disease in dogs with acute, severe thoracolumbar myelopathy secondary to Type I IVDH.AnimalsThirty-nine client-owned dogs with thoracolumbar myelopathy secondary to IVDH.MethodsProspective controlled cohort study. Serum was collected from dogs undergoing hemilaminectomy at multiple timepoints. Final neurological status was established at 12 months and groups were stratified accordingly. Comparisons between outcome and pNF-H concentration at each timepoint was examined using Kruskal-Wallis analysis of variance on ranks and receiver operator characteristics curve analysis.ResultsMedian serum pNF-H concentrations were not significantly different between deep pain negative dogs that did or did not recover at any timepoint (baseline: 0.37 ng/mL [0-0.9 ng/mL] vs 0 ng/mL [0-0.9 ng/mL], P > 1; 24 hours: 1.25 ng/mL [0.35-7.23 ng/mL] vs 1.53 ng/mL [0-11.94 ng/mL], P > 1; 48 hours: 1.22 ng/mL [0.63-6.62 ng/mL] vs 2.12 ng/mL [0-20.72 ng/mL], P >  1; 72 hours: 2.77 ng/mL [1.33-6.62 ng/mL] vs 16.69 ng/mL [4.02-40.12 ng/mL], P >  1). Dogs that developed PMM had significantly higher serum pNF-H concentrations after surgery compared to all other cohorts at 24 hours: 39.88 ng/mL (25.74-50.68 ng/mL); P < .05 and 72 hours: 223.9 ng/mL (155.4-263.7 ng/mL); P < .05. A serum pNF-H concentration ≥31.39 ng/mL was 83.33% sensitive and 100% specific for identifying PMM in this cohort.Conclusions and clinical importanceSerum pNF-H is a promising biomarker for antemortem diagnosis of PMM in dogs with acute, severe thoracolumbar myelopathy secondary to Type I IVDH

Lumbar spinal cord microglia exhibited increased activation in aging dogs compared with young adult dogs.

Altered microglia function contributes to loss of CNS homeostasis during aging in the brain. Few studies have evaluated age-related alterations in spinal cord microglia. We previously demonstrated that lumbar spinal cord microglial expression of inducible nitric oxide synthase (iNOS) was equivalent between aging, neurologically normal dogs and dogs with canine degenerative myelopathy (Toedebusch et al. 2018, Mol Cell Neurosci. 88, 148-157). This unexpected finding suggested that microglia in aging spinal cord have a pro-inflammatory polarization. In this study, we reexamined our microglial results (Toedebusch et al. 2018, Mol Cell Neurosci. 88, 148-157) within the context of aging rather than disease by comparing microglia in aging versus young adult dogs. For both aging and young adult dogs, the density of microglia was significantly higher closest to the motor neuron cell body. However, there was no difference in densities between aging versus young adult dogs at all distances except for the furthest distance analyzed. The number of motor neurons with polarized microglia was higher in aging dogs; yet, the density per motor neuron of arginase-1-expressing microglia was reduced in aging dogs compared with young adult dogs. Finally, aging dogs had increased steady-state mRNA levels for genes consistent with activated microglia compared with young adult dogs. However, altered mRNA levels were limited to the lumbar spinal cord. These data suggested that aging dog spinal cord microglia exhibit regional immunophenotypic differences, which may render lumbar motor neurons more susceptible to age-related pathological insults

Longevity and mortality in cats: A single institution necropsy study of 3108 cases (1989–2019)

Client-owned cats who underwent a post-mortem examination (n = 3,108) at a veterinary medical teaching hospital between 1989 and 2019 were studied to determine longevity and factors affecting mortality. Demographic factors, environmental factors, age, and causes of death were assessed. Sexes included 5.66% intact females, 39.86% spayed females, 6.95% intact males and 47.49% neutered males. 84.2% were mixed breed cats. Age at death was known for 2,974 cases with a median of 9.07 years. Cancer was the most common pathophysiologic cause of death (35.81%) and was identified in 41.3% of cats. When categorized by organ system, mortality was most attributed to multiorgan/systemic (21.72%). Renal histologic abnormalities were noted in 62.84% of cats but was considered the primary cause of death in only 13.06% of cats. Intact female and male cats had significantly shorter lifespans than their spayed or neutered counterparts. FeLV positive status was associated with decreased longevity (P<0.0001) while FIV status was not. This study reports on risk factors associated with mortality and highlights areas of research that may contribute to improved lifespan in cats