Early Signs of Neuroinflammation in the Postnatal Wobbler Mouse Model of Amyotrophic Lateral Sclerosis.

The Wobbler mouse is an accepted model of sporadic amyotrophic lateral sclerosis. The spinal cord of clinically symptomatic animals (3-5 months old) shows vacuolar motoneuron degeneration, inflammation, and gliosis accompanied by motor impairment. However, data are not conclusive concerning pathological changes appearing early after birth. To answer this question, we used postnatal day (PND) 6 genotyped Wobbler pups to determine abnormalities of glia and neurons at this early age period in the spinal cord. We found astrogliosis, microgliosis with morphophenotypic changes pointing to active ameboid microglia, enhanced expression of the proinflammatory markers TLR4, NFkB, TNF, and inducible nitric oxide synthase. The astrocytic enzyme glutamine synthase and the glutamate-aspartate transporter GLAST were also reduced in PND 6 Wobbler pups, suggesting excitotoxicity due to impaired glutamate homeostasis. At the neuronal level, PND 6 Wobblers showed swollen soma, increased choline acetyltransferase immunofluorescence staining, and low expression of the neuronal nuclear antigen NeuN. However, vacuolated motoneurons, a typical signature of older clinically symptomatic Wobbler mice, were absent in the spinal cord of PND 6 Wobblers. The results suggest predominance of neuroinflammation and abnormalities of microglia and astrocytes at this early period of Wobbler life, accompanied by some neuronal changes. Data support the non-cell autonomous hypothesis of the Wobbler disorder, and bring useful information with regard to intervening molecular inflammatory mechanisms at the beginning stage of human motoneuron degenerative diseases.

Inhibition of sphingolipid synthesis improves outcomes and survival in GARP mutant wobbler mice, a model of motor neuron degeneration.

Numerous mutations that impair retrograde membrane trafficking between endosomes and the Golgi apparatus lead to neurodegenerative diseases. For example, mutations in the endosomal retromer complex are implicated in Alzheimer's and Parkinson's diseases, and mutations of the Golgi-associated retrograde protein (GARP) complex cause progressive cerebello-cerebral atrophy type 2 (PCCA2). However, how these mutations cause neurodegeneration is unknown. GARP mutations in yeast, including one causing PCCA2, result in sphingolipid abnormalities and impaired cell growth that are corrected by treatment with myriocin, a sphingolipid synthesis inhibitor, suggesting that alterations in sphingolipid metabolism contribute to cell dysfunction and death. Here we tested this hypothesis in wobbler mice, a murine model with a homozygous partial loss-of-function mutation in Vps54 (GARP protein) that causes motor neuron disease. Cytotoxic sphingoid long-chain bases accumulated in embryonic fibroblasts and spinal cords from wobbler mice. Remarkably, chronic treatment of wobbler mice with myriocin markedly improved their wellness scores, grip strength, neuropathology, and survival. Proteomic analyses of wobbler fibroblasts revealed extensive missorting of lysosomal proteins, including sphingolipid catabolism enzymes, to the Golgi compartment, which may contribute to the sphingolipid abnormalities. Our findings establish that altered sphingolipid metabolism due to GARP mutations contributes to neurodegeneration and suggest that inhibiting sphingolipid synthesis might provide a useful strategy for treating these disorders.

Progesterone and Allopregnanolone Neuroprotective Effects in the Wobbler Mouse Model of Amyotrophic Lateral Sclerosis.

Progesterone regulates a number of processes in neurons and glial cells not directly involved in reproduction or sex behavior. Several neuroprotective effects are better observed under pathological conditions, as shown in the Wobbler mouse model of amyotrophic laterals sclerosis (ALS). Wobbler mice are characterized by forelimb atrophy due to motoneuron degeneration in the spinal cord, and include microgliosis and astrogliosis. Here we summarized current evidence on progesterone reversal of Wobbler neuropathology. We demonstrated that progesterone decreased motoneuron vacuolization with preservation of mitochondrial respiratory complex I activity, decreased mitochondrial expression and activity of nitric oxide synthase, increased Mn-dependent superoxide dismutase, stimulated brain-derived neurotrophic factor, increased the cholinergic phenotype of motoneurons, and enhanced survival with a concomitant decrease of death-related pathways. Progesterone also showed differential effects on glial cells, including increased oligodendrocyte density and downregulation of astrogliosis and microgliosis. These changes associate with reduced anti-inflammatory markers. The enhanced neurochemical parameters were accompanied by longer survival and increased muscle strength in tests of motor behavior. Because progesterone is locally metabolized to allopregnanolone (ALLO) in nervous tissues, we also studied neuroprotection by this derivative. Treatment of Wobbler mice with ALLO decreased oxidative stress and glial pathology, increased motoneuron viability and clinical outcome in a progesterone-like manner, suggesting that ALLO could mediate some progesterone effects in the spinal cord. In conclusion, the beneficial effects observed in different parameters support the versatile properties of progesterone and ALLO in a mouse model of motoneuron degeneration. The studies foresee future therapeutic opportunities with neuroactive steroids for deadly diseases like ALS.

Magnetic resonance imaging and neurological findings in dogs with disc-associated cervical spondylomyelopathy: a case series.

BACKGROUND: Canine cervical spondylomyelopathy can be separated into osseous and disc-associated (DA-CSM) forms. Our aim was to describe the magnetic resonance imaging (using a high-field scanner) and neurological findings in dogs with DA-CSM and investigate a relationship between these findings. RESULTS: Sixty-three dogs were included: 60/63 (95 %) were large breeds, with Doberman Pinschers and males over-represented (70 %). Mean and median age at the time of diagnosis was 7.25 and 7.2 years (range 0.41-12 years). Chronic signs were noted in 52/63 (83 %) dogs, with proprioceptive ataxia the most common. Main site of spinal cord compression was commonly C6-7 or C5-6. Thirty-six (57 %) dogs had various sites of spinal cord compression. Most dogs younger than 6 years of age had a single affected site. Foraminal stenosis was present in 51/63 dogs (81 %). T2-weighted hyperintensity was present in 40/63 dogs (63 %). 88 % of the articular processes showed degenerative changes, which correlated strongly with intervertebral disc degeneration. Ligamentum flavum hypertrophy was seen in 38 % of dogs. No correlation was observed between neurologic signs and number of affected sites. A moderate positive correlation was observed between severity of spinal cord compression and neurologic grade (r 0.48; p < 0.001). CONCLUSIONS: DA-CSM was predominantly observed in older, male Dobermans, with lesions located in the caudal cervical vertebral region. It was also seen in dogs 3 years of age or even younger (8 %). Single compressive lesions were more common in dogs younger than 6 years of age. Many dogs had concomitant changes (e.g.: ligamentum flavum hypertrophy and foraminal stenosis). Most dogs with ligamentum flavum hypertrophy were 6 years or older. A positive correlation was observed between severity of spinal cord compression and neurologic grade, but multilevel compression was not associated with more severe neurologic signs. A very high percentage of dogs had articular process degenerative changes. Possible biomechanical or genetic relationships between degenerative changes in articular processes, ligamentum flavum, and intervertebral discs warrants further investigation.

Little Helpers or Mean Rogue-Role of Microglia in Animal Models of Amyotrophic Lateral Sclerosis.

Amyotrophic lateral sclerosis (ALS) is one of the most common neurodegenerative diseases, causing degeneration of both upper and lower motor neurons in the central nervous system (CNS). ALS patients suffer from hyperreflexia, spasticity, paralysis and muscle atrophy and typically die due to respiratory failure 1-5 years after disease onset. In addition to the degeneration of motor neurons on the cellular level, ALS has been associated with neuroinflammation, such as microgliosis. Microglial activation in ALS can either be protective or degenerative to the neurons. Among others, mutations in superoxide dismutase 1 (SOD1), chromosome 9 open reading frame 72 (C9Orf72), transactive response DNA binding protein (TDP) 43 and vacuolar protein sorting-associated protein 54 (VPS54) genes have been associated with ALS. Here, we describe the dual role and functionality of microglia in four different in vivo ALS models and search for the lowest common denominator with respect to the role of microglia in the highly heterogeneous disease of ALS.

Association of neurologic signs with high-field MRI findings in 100 dogs with osseous-associated cervical spondylomyelopathy.

Although osseous-associated cervical spondylomyelopathy (OA-CSM) findings have been well described using magnetic resonance imaging (MRI), there are no large-scale published studies on the associations between dog size, age, high-field MRI and neurologic findings. Using a retrospective, observational study design, we aimed to investigate an association between neurologic and high-field MRI characteristics in OA-CSM. Records were reviewed for dogs diagnosed with OA-CSM using high-field MRI. One-hundred dogs were included: 73/100 (73%) were giant breeds, 27/100 (27%) large breeds. Mean and median ages were, respectively, 3.1 and 2 years (0.3-9.75 years), with 2.6 and 2 years for giant-breed; and 4.4 and 4 years for large-breed dogs. The majority of dogs were male (75%) with chronic presentation (89%), more than one site of spinal cord compression (78%) and foraminal stenosis (91%). Dogs with multiples sites of spinal cord compression were more likely to have severe spinal cord compression (p < 0.001), severe foraminal stenosis (p < 0.001) and ligamentum flavum/soft tissue proliferation (p = 0.03) than those with a single compressive site. There was a weak correlation between neurologic grade and severity of spinal cord compression (r = 0.27; p = 0.007), number of affected sites (r = 0.24; p = 0.0183) and spinal cord T2W hyperintensity (r = 0.24; p = 0.0152). Intervertebral disc degeneration was seen in 80% of dogs. Age did not appear to have a prominent role in the manifestation of OA-CSM. This study showed that OA-CSM affects a sizeable proportion of young large-breed, in addition to giant-breed dogs.

Insights into the Therapeutic Potential of Glucocorticoid Receptor Modulators for Neurodegenerative Diseases.

Glucocorticoids are crucial for stress-coping, resilience, and adaptation. However, if the stress hormones become dysregulated, the vulnerability to stress-related diseases is enhanced. In this brief review, we discuss the role of glucocorticoids in the pathogenesis of neurodegenerative disorders in both human and animal models, and focus in particular on amyotrophic lateral sclerosis (ALS). For this purpose, we used the Wobbler animal model, which mimics much of the pathology of ALS including a dysfunctional hypothalamic-pituitary-adrenal axis. We discuss recent studies that demonstrated that the pathological cascade characteristic for motoneuron degeneration of ALS is mimicked in the genetically selected Wobbler mouse and can be attenuated by treatment with the selective glucocorticoid receptor antagonist (GRA) CORT113176. In long-term treatment (3 weeks) GRA attenuated progression of the behavioral, inflammatory, excitatory, and cell-death-signaling pathways while increasing the survival signal of serine-threonine kinase (pAkt). The action mechanism of the GRA may be either by interfering with GR deactivation or by restoring the balance between pro- and anti-inflammatory signaling pathways driven by the complementary mineralocorticoid receptor (MR)- and GR-mediated actions of corticosterone. Accordingly, GR antagonism may have clinical relevance for the treatment of neurodegenerative diseases.

The microRNA miR-375-3p and the Tumor Suppressor NDRG2 are Involved in Sporadic Amyotrophic Lateral Sclerosis.

BACKGROUND/AIMS: Amyotrophic lateral sclerosis (ALS) is the most common degenerative motor neuron disease in humans. However, the pathogenesis of ALS is not yet understood. The wobbler mouse is considered as an animal model for the sporadic form of ALS due to its spontaneous mutation in the Vps54 gene. Due to transactivation of NDRG2 by p53, this tumor suppressor might play a functional role in stress induced cell death in wobbler mice as well as ALS patients. Furthermore, deregulated microRNAs are often related to neurodegenerative diseases. Thus, the NDRG2 linked miR-375-3p was of interest for this study. METHODS: Here, we investigated the relevance of NDRG2 and miR-375-3p for the pathomechanism of the motor neuronal degeneration in wobbler mice by investigating expression level via qPCR and Western Blot as well as localization of these molecules in the cervical spinal cord by in situ hybridization, immunostaining and mass spectrometric analysis. RESULTS: We were able to show a differential regulation of the expression of NDRG2 as well as miR-375-3p in the cervical part of the spinal cord of wobbler mice. In addition, for the first time we were able to demonstrate an expression of NDRG2 in motor neurons using different techniques. CONCLUSION: The present study has shown NDRG2 and miR-375-3p to be promising targets for further research of the pathogenesis of sporadic ALS in the wobbler mouse model. Based on these results and in combination with previous published data we could develop a putative pro-apoptotic mechanism in the spinal cord of the wobbler mouse.

Update on Surgical Treatment of Wobblers.

Cervical vertebral compressive myelopathy (CVCM) represents the most significant disease of the spinal cord in horses for which surgical treatment is described. Current surgical methods used include ventral interbody fusion with kerf cut cylinders and dorsal laminectomy. Polyaxial pedicle screw and rod constructs and ventral locking compression plating have been introduced in the treatment of equine CVCM and present promising alternative approaches to achieve ventral interbody fusion. Advancements in diagnostic imaging and endoscopy of the cervical vertebral canal may improve reliable preoperative identification of the exact locations of spinal cord compression in horses with CVCM to improve postoperative outcomes.

Comparison of angle, shape, and position of articular processes in Dobermans and Great Danes with and without cervical spondylomyelopathy.

BACKGROUND: Cervical spondylomyelopathy (CSM), also known as wobbler syndrome, affects mainly large and giant-breed dogs, causing compression of the cervical spinal cord and/or nerve roots. Structural and dynamic components seem to play a role in the development of CSM; however, pathogenesis is not yet fully understood. Physiologic and pathologic movements of the cervical spine depend on the morphology and morphometry of articular processes, as well as on intervertebral discs and vertebral column ligaments. Moreover, the characteristics of the articular processes affect motion and stability of the vertebral column. The goal of this study was to investigate the angle, shape, and position of the articular surfaces within the articular processes and compare them between Doberman Pinschers and Great Danes with and without cervical spondylomyelopathy. RESULTS: Magnetic resonance images were obtained for 60 dogs: 15 clinically normal Dobermans (Dob-N), 15 CSM-affected Dobermans (Dob-CSM), 15 clinically normal Great Danes (GD-N), and 15 CSM-affected Great Danes (GD-CSM). Angle, shape, and position (lateral distance) of the articular surfaces from the articular processes were analyzed from C2-3 to C7-T1. Results indicate that the mean angle was different between Dob-CSM and GD-CSM at C4-5, C5-6, and C6-7, and between GD-N and GD-CSM at C6-7. There were differences between Dob-N and GD-N, and between Dob-CSM and GD-CSM for the lateral distance at most locations, except C2-3. Compared with Great Danes, Dobermans generally had a greater proportion of concave caudal surfaces at C4-5, C5-6, and C6-7. Concave articular surfaces have been associated with greater axial rotation. This may explain the high proportion of disc-associated CSM in Dobermans compared to Great Danes. The differences between breeds suggest they may have different motion patterns in the caudal cervical vertebral column. CONCLUSIONS: Considering that no differences in angle, shape, or position of the articular surfaces within the articular processes were found between normal and CSM-affected dogs, their relevance appears to have a secondary role in the pathogenesis of CSM.

Evaluation of osseous-associated cervical spondylomyelopathy in dogs using kinematic magnetic resonance imaging.

Osseous-associated cervical spondylomyelopathy in dogs is characterized by both static and dynamic spinal cord compression; however, standard MRI methods only assess static compression. In humans with cervical spondylotic myelopathy, kinematic MRI is commonly used to diagnose dynamic spinal cord compressions. The purpose of this prospective, analytical study was to evaluate kinematic MRI as a method for characterizing the dynamic component of osseous-associated cervical spondylomyelopathy in dogs. We hypothesized that kinematic MRI would allow visualization of spinal cord compressions that were not identified with standard imaging. Twelve client-owned dogs with osseous-associated cervical spondylomyelopathy were enrolled. After standard MRI confirmed a diagnosis of osseous-associated cervical spondylomyelopathy, a positioning device was used to perform additional MRI sequences with the cervical vertebral column flexed and extended. Morphologic and morphometric (spinal cord height, intervertebral disc width, spinal cord width, vertebral canal height, and spinal cord area) assessments were recorded for images acquired with neutral, flexion, and extension imaging. A total of 25 compressions were seen with neutral positioning, while extension identified 32 compressions. There was a significant association between extension positioning and presence of a compressive lesion at C4-C5 (p = 0.02). Extension was also associated with a change in the most severe site of compression in four out of 12 (33%) dogs. None of the patients deteriorated neurologically after kinematic imaging. We concluded that kinematic MRI is a feasible method for evaluating dogs with osseous-associated cervical spondylomyelopathy, and can reveal new compressions not seen with neutral positioning.

Cervical Vertebral Lesions in Equine Stenotic Myelopathy.

Skeletal lesions in the articular processes of cervical vertebrae C2 to C7 were compared between Thoroughbred horses with cervical stenotic myelopathy (17 males, 2 females; age, 6-50 months) and controls (6 males, 3 females; age, 9-67 months). Lesions identified by magnetic resonance imaging occurred with an increased frequency and severity in diseased horses and were not limited to sites of spinal cord compression. Lesions involved both the articular cartilage and trabecular bone and were further characterized using micro-computed tomography and histopathology. The most common histologic lesions included osteochondrosis, osseous cyst-like structures, fibrous tissue replacement of trabecular bone, retained cartilage matrix spicules, and osteosclerosis. Osseous cyst-like structures were interpreted to be true bone cysts given they were a closed cavity with a cellular lining that separated the cyst from surrounding bone. This is the first report of bone cysts in the cervical articular processes of horses with cervical stenotic myelopathy. The morphology and distribution of the lesions provide additional support for the previously proposed pathogenesis that developmental abnormalities with likely secondary biomechanical influences on the cervical spine contribute to equine cervical stenotic myelopathy.

Neuroprotective effects of the Sigma-1 receptor (S1R) agonist PRE-084, in a mouse model of motor neuron disease not linked to SOD1 mutation.

The identification of novel molecular targets crucially involved in motor neuron degeneration/survival is a necessary step for the development of hopefully more effective therapeutic strategies for amyotrophic lateral sclerosis (ALS) patients. In this view, S1R, an endoplasmic reticulum (ER)-resident receptor with chaperone-like activity, has recently attracted great interest. S1R is involved in several processes leading to acute and chronic neurodegeneration, including ALS pathology. Treatment with the S1R agonist PRE-084 improves locomotor function and motor neuron survival in presymptomatic and early symptomatic mutant SOD1-G93A ALS mice. Here, we tested the efficacy of PRE-084 in a model of spontaneous motor neuron degeneration, the wobbler mouse (wr) as a proof of concept that S1R may be regarded as a key therapeutic target also for ALS cases not linked to SOD1 mutation. Increased staining for S1R was detectable in morphologically spared cervical spinal cord motor neurons of wr mice both at early (6th week) and late (12th week) phases of clinical progression. S1R signal was also detectable in hypertrophic astrocytes and reactive microglia of wr mice. Chronic treatment with PRE-084 (three times a week, for 8weeks), starting at symptom onset, significantly increased the levels of BDNF in the gray matter, improved motor neuron survival and ameliorated paw abnormality and grip strength performance. In addition, the treatment significantly reduced the number of reactive astrocytes whereas, that of CD11b+ microglial cells was increased. A deeper evaluation of microglial markers revealed significant increased number of cells positive for the pan-macrophage marker CD68 and of CD206+ cells, involved in tissue restoration, in the white matter of PRE-084-treated mice. The mRNA levels of TNF-α and IL-1ß were not affected by PRE-084 treatment. Thus, our results support pharmacological manipulation of S1R as a promising strategy to cure ALS and point to increased availability of growth factors and modulation of astrocytosis and of macrophage/microglia as part of the mechanisms involved in S1R-mediated neuroprotection.

Comparison of noncontrast computed tomography and high-field magnetic resonance imaging in the evaluation of Great Danes with cervical spondylomyelopathy.

Computed tomography (CT) provides excellent bony detail, whereas magnetic resonance (MR) imaging is superior in evaluating the neural structures. The purpose of this prospective study was to assess interobserver and intermethod agreement in the evaluation of cervical vertebral column morphology and lesion severity in Great Danes with cervical spondylomyelopathy by use of noncontrast CT and high-field MR imaging. Fifteen client-owned affected Great Danes were enrolled. All dogs underwent noncontrast CT under sedation and MR imaging under general anesthesia of the cervical vertebral column. Three observers independently evaluated the images to determine the main site of spinal cord compression, direction and cause of the compression, articular process joint characteristics, and presence of foraminal stenosis. Overall intermethod agreement, intermethod agreement for each observer, overall interobserver agreement, and interobserver agreement between pairs of observers were calculated by use of kappa (κ) statistics. The highest overall intermethod agreements were obtained for the main site of compression and direction of compression with substantial agreements (κ = 0.65 and 0.62, respectively), whereas the lowest was obtained for right-sided foraminal stenosis (κ = 0.39, fair agreement). For both imaging techniques, the highest and lowest interobserver agreements were recorded for the main site of compression and degree of articular joint proliferation, respectively. While different observers frequently agree on the main site of compression using both imaging techniques, there is considerable variation between modalities and among observers when assessing articular process characteristics and foraminal stenosis. Caution should be exerted when comparing image interpretations from multiple observers.

Evaluation of traditional and novel radiographic vertebral ratios in Great Danes with versus without cervical spondylomyelopathy.

Great Danes are predisposed to osseous-associated cervical spondylomyelopathy (Wobbler syndrome). The first aim of this prospective study was to compare values measured using previously published intravertebral and intervertebral ratio methods and a novel ventrodorsal ratio method in radiographs of clinically normal and affected Great Danes. The second aim was to determine whether these ratios could be used as predictors of sites of spinal cord compression based on magnetic resonance imaging (MRI). Thirty dogs (15 normal, 15 affected) were prospectively enrolled. Lateral and ventrodorsal radiographs were obtained and six measurements were recorded from C3-T1. For each vertebral location, intravertebral ratios and intervertebral ratios were calculated from lateral views, and the ratio of the distance between the articular process joints vs. vertebral body width (novel ventrodorsal ratio) was calculated from ventrodorsal views. Values for these three ratios were compared, by vertebral location and dog group. Intravertebral and intervertebral ratios did not differ between dog groups. The ventrodorsal ratio was significantly smaller in affected Great Danes at C5-6 (P = 0.005) and C6-7 (P < 0.001). The ventrodorsal ratio was significantly associated with MRI presence of spinal cord compression. For each 0.1 unit increase in this ratio value, there was a 65% decrease in the odds of spinal cord compression being present at that site, independent of vertebral location (P = 0.002). Findings from this study supported use of the novel ventrodorsal ratio as an initial radiographic screening method for Great Danes with suspected cervical spondylomyelopathy.

Testosterone Reduces Myelin Abnormalities in the Wobbler Mouse Model of Amyotrophic Lateral Sclerosis.

Amyotrophic lateral sclerosis (ALS) is a fatal motoneuron degenerative disease that is associated with demyelination. The Wobbler (WR) mouse exhibits motoneuron degeneration, gliosis and myelin deterioration in the cervical spinal cord. Since male WRs display low testosterone (T) levels in the nervous system, we investigated if T modified myelin-relative parameters in WRs in the absence or presence of the aromatase inhibitor, anastrozole (A). We studied myelin by using luxol-fast-blue (LFB) staining, semithin sections, electron microscopy and myelin protein expression, density of IBA1+ microglia and mRNA expression of inflammatory factors, and the glutamatergic parameters glutamine synthetase (GS) and the transporter GLT1. Controls and WR + T showed higher LFB, MBP and PLP staining, lower g-ratios and compact myelin than WRs and WR + T + A, and groups showing the rupture of myelin lamellae. WRs showed increased IBA1+ cells and mRNA for CD11b and inflammatory factors (IL-18, TLR4, TNFαR1 and P2Y12R) vs. controls or WR + T. IBA1+ cells, and CD11b were not reduced in WR + T + A, but inflammatory factors' mRNA remained low. A reduction of GS+ cells and GLT-1 immunoreactivity was observed in WRs and WR + T + A vs. controls and WR + T. Clinically, WR + T but not WR + T + A showed enhanced muscle mass, grip strength and reduced paw abnormalities. Therefore, T effects involve myelin protection, a finding of potential clinical translation.

Elevated NLRP3 Inflammasome Activation Is Associated with Motor Neuron Degeneration in ALS.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by motor neuron degeneration in the central nervous system. Recent research has increasingly linked the activation of nucleotide oligomerization domain-like receptor protein 3 (NLRP3) inflammasome to ALS pathogenesis. NLRP3 activation triggers Caspase 1 (CASP 1) auto-activation, leading to the cleavage of Gasdermin D (GSDMD) and pore formation on the cellular membrane. This process facilitates cytokine secretion and ultimately results in pyroptotic cell death, highlighting the complex interplay of inflammation and neurodegeneration in ALS. This study aimed to characterize the NLRP3 inflammasome components and their colocalization with cellular markers using the wobbler mouse as an ALS animal model. Firstly, we checked the levels of miR-223-3p because of its association with NLRP3 inflammasome activity. The wobbler mice showed an increased expression of miR-223-3p in the ventral horn, spinal cord, and cerebellum tissues. Next, increased levels of NLRP3, pro-CASP 1, cleaved CASP 1 (c-CASP 1), full-length GSDMD, and cleaved GDSMD revealed NLRP3 inflammasome activation in wobbler spinal cords, but not in the cerebellum. Furthermore, we investigated the colocalization of the aforementioned proteins with neurons, microglia, and astrocyte markers in the spinal cord tissue. Evidently, the wobbler mice displayed microgliosis, astrogliosis, and motor neuron degeneration in this tissue. Additionally, we showed the upregulation of protein levels and the colocalization of NLRP3, c-CASP1, and GSDMD in neurons, as well as in microglia and astrocytes. Overall, this study demonstrated the involvement of NLRP3 inflammasome activation and pyroptotic cell death in the spinal cord tissue of wobbler mice, which could further exacerbate the motor neuron degeneration and neuroinflammation in this ALS mouse model.

Magnetic resonance imaging and neurologic characterization of combined osseous- and disc-associated cervical spondylomyelopathy in dogs.

BACKGROUND: Although both disc- or osseous-associated forms of cervical spondylomyelopathy (CSM) are observed in the same dogs, this combined form has not been thoroughly evaluated. OBJECTIVES: To describe imaging characteristics of dogs with concurrent disc- and osseous CSM and investigate an association between findings on neurological examination and imaging. ANIMALS: Sixty dogs with disc and osseous-associated CSM from 232 CSM-affected dogs. METHODS: Retrospective study. Dogs diagnosed via high-field MRI with a combination of intervertebral disc (IVD) protrusion and osseous proliferation of articular processes, dorsal lamina, or both were identified. Large and giant breed dogs were grouped according to whether combined compressions were at the same site or different sites. Statistical methods were used to investigate the association and relationship between variables. RESULTS: Thirty-five out of 60 (58%) were large breeds and 22/60 (37%) were giant breeds. Mean and median age was 6.6 and 7 years respectively (range, 0.75-11 years). Forty of the 60 dogs (67%) had concurrent osseous and disc-associated spinal cord compression in the same location. This was considered the main compression site in 32/40 (80%) dogs. Dogs with osseous- and disc-associated compressions at the same site were more likely to have a higher neurologic grade (P = .04). CONCLUSIONS AND CLINICAL IMPORTANCE: A substantial percentage of dogs with CSM present with concomitant IVD protrusion and osseous proliferations, most at the same site. Characterizing this combined form is important in the management of dogs with CSM because it could affect treatment choices.

Instrumented cervical fusion using patient specific end-plate conforming interbody devices with a micro-porous structure in nine dogs with disk-associated cervical spondylomyelopathy.

Objective: To report the medium and long-term outcome of nine dogs with disk-associated cervical spondylomyelopathy (DA-CSM), treated by instrumented interbody fusion using patient specific end-plate conforming device that features a micro-porous structure to facilitate bone in-growth. Study design: A retrospective clinical study. Animals: Nine medium and large breed dogs. Methods: Medical records at two institutions were reviewed between January 2020 and 2023. Following magnetic resonance imaging (MRI) diagnosis of DA-CSM, pre-operative computed tomography (CT) scans were exported to computer software for in-silico surgical planning. Interbody devices were 3D-manufactured by selecting laser melting in titanium alloy. These were surgically implanted at 13 segments alongside mono-or bi-cortical vertebral stabilization systems. Follow-up included neurologic scoring and CT scans post-operative, at medium-term follow up and at long-term follow-up where possible. Interbody fusion and implant subsidence were evaluated from follow-up CT scans. Results: Nine dogs were diagnosed with DA-CSM between C5-C7 at a total of 13 operated segments. Medium-term follow up was obtained between 2 and 8 months post-operative (3.00 ± 1.82 months). Neurologic scoring improved (p = 0.009) in eight of nine dogs. Distraction was significant (p < 0.001) at all segments. Fusion was evident at 12/13 segments. Subsidence was evident at 3/13 operated segments but was only considered clinically relevant in one dog that did not improve; as clinical signs were mild, revision surgery was not recommended. Long-term follow up was obtained between 9 and 33 months (14.23 ± 8.24 months); improvement was sustained in 8 dogs. The dog that suffered worsened thoracic limb paresis at medium-term follow up was also diagnosed with immune-mediated polyarthropathy (IMPA) and was euthanased 9 months post-operative due to unacceptable side-effects of corticosteroid therapy. Conclusion: End-plate conforming interbody devices with a micro-porous structure were designed, manufactured, and successfully implanted in dog with DA-CSM. This resulted in CT-determined fusion with minimal subsidence in the majority of operated segments. Clinical significance: The technique described can be used to distract and fuse cervical vertebrae in dogs with DA-CSM, with favorable medium-and long-term outcomes.

Defects in Glutathione System in an Animal Model of Amyotrophic Lateral Sclerosis.

Amyotrophic lateral sclerosis (ALS) is a progredient neurodegenerative disease characterized by a degeneration of the first and second motor neurons. Elevated levels of reactive oxygen species (ROS) and decreased levels of glutathione, which are important defense mechanisms against ROS, have been reported in the central nervous system (CNS) of ALS patients and animal models. The aim of this study was to determine the cause of decreased glutathione levels in the CNS of the ALS model wobbler mouse. We analyzed changes in glutathione metabolism in the spinal cord, hippocampus, cerebellum, liver, and blood samples of the ALS model, wobbler mouse, using qPCR, Western Blot, HPLC, and fluorometric assays. Here, we show for the first time a decreased expression of enzymes involved in glutathione synthesis in the cervical spinal cord of wobbler mice. We provide evidence for a deficient glutathione metabolism, which is not restricted to the nervous system, but can be seen in various tissues of the wobbler mouse. This deficient system is most likely the reason for an inefficient antioxidative system and, thus, for elevated ROS levels.