Toll-like receptors 4 and 9 are responsible for the maintenance of the inflammatory reaction in canine steroid-responsive meningitis-arteritis, a large animal model for neutrophilic meningitis

BACKGROUND: Steroid-responsive meningitis-arteritis (SRMA) is a systemic inflammatory disease affecting young adult dogs and a potential large animal model for neutrophilic meningitis. Similarities between SRMA and infectious central nervous system (CNS) diseases in lymphocyte subsets suggest an infectious origin. Toll-like receptors (TLRs) are pattern recognition receptors playing an important role in innate immunity. Due to their ability to recognize both self and non-self antigens, we hypothesize that TLRs are among the key factors for the induction of the inflammatory process in SRMA and provide an indirect hint on the etiology of the disease. METHODS: The expression profile of cell surface TLRs (TLR2, TLR4 and TLR5) and intracellular TLRs (TLR3 and TLR9) of canine leukocytes was analyzed by immunophenotyping and subsequent flow cytometric measurements. Experiments were performed on cerebrospinal fluid (CSF) and peripheral blood (PB) samples of dogs affected with SRMA during the acute phase (n = 14) as well as during treatment (n = 23) and compared with those of dogs with bacterial meningitis (n = 3), meningoencephalitis of unknown etiology (n = 6), neoplasia of the central nervous system (n = 6) and a group of dogs with miscellaneous neurological diseases (n = 9). Two additional control groups consisted of dogs with pyogenic infections (n = 13) and of healthy dogs (n = 6). RESULTS: All examined groups showed a high percentage of TLR2, TLR4 and TLR5 positive PB polymorphonuclear cells (PMNs) in comparison to healthy dogs. Very high values of TLR9 positive PB PMNs were detected in acute SRMA. Only a few similarities were found between SRMA patients and dogs with pyogenic infections, both groups were characterized by high expression of TLR4 positive PB monocytes. Glucocorticosteroid therapy reduced TLR2, TLR4 and TLR9 expression in PB monocytes. CONCLUSIONS: A relatively high expression of TLR4 and TLR9 in acute SRMA suggests that these two receptors might be involved in the inflammatory process in SRMA, enhancing the autoimmune reaction. Systematic CSF cell analysis for TLRs can be performed in future treatment studies in larger animals, such as dogs

Machine learning predicts histologic type and grade of canine gliomas based on MRI texture analysis.

Conventional MRI features of canine gliomas subtypes and grades significantly overlap. Texture analysis (TA) quantifies image texture based on spatial arrangement of pixel intensities. Machine learning (ML) models based on MRI-TA demonstrate high accuracy in predicting brain tumor types and grades in human medicine. The aim of this retrospective, diagnostic accuracy study was to investigate the accuracy of ML-based MRI-TA in predicting canine gliomas histologic types and grades. Dogs with histopathological diagnosis of intracranial glioma and available brain MRI were included. Tumors were manually segmented across their entire volume in enhancing part, non-enhancing part, and peri-tumoral vasogenic edema in T2-weighted (T2w), T1-weighted (T1w), FLAIR, and T1w postcontrast sequences. Texture features were extracted and fed into three ML classifiers. Classifiers' performance was assessed using a leave-one-out cross-validation approach. Multiclass and binary models were built to predict histologic types (oligodendroglioma vs. astrocytoma vs. oligoastrocytoma) and grades (high vs. low), respectively. Thirty-eight dogs with a total of 40 masses were included. Machine learning classifiers had an average accuracy of 77% for discriminating tumor types and of 75.6% for predicting high-grade gliomas. The support vector machine classifier had an accuracy of up to 94% for predicting tumor types and up to 87% for predicting high-grade gliomas. The most discriminative texture features of tumor types and grades appeared related to the peri-tumoral edema in T1w images and to the non-enhancing part of the tumor in T2w images, respectively. In conclusion, ML-based MRI-TA has the potential to discriminate intracranial canine gliomas types and grades

Feasability of a Frameless Brain Biopsy System for Companion Animals Using Cone-Beam CT-Based Automated Registration.

The aim of the present study was to evaluate the use of a novel intraoperative cone-beam computed tomography (CBCT)-based automated registration system for frameless stereotactic brain biopsy in companion animals. An experimental cadaveric study evaluated thalamic and piriform lobe target site needle placement error in three dogs and three cats without a history of intracranial disease. Diagnostic accuracy and diagnostic yield were prospectively evaluated in twenty-four client-owned dogs and four cats with intracranial disease. Twenty-one procedures were performed post mortem (eighteen dogs and three cats), and seven biopsy procedures were performed in alive patients (six dogs and one cat). Procedural duration was evaluated in ten post mortem and four living patients. Outcome was evaluated in six dogs and one cat. In dogs, the calculated median needle placement error was 1.8 mm (range 0.71-2.84 mm) and 1.53 mm (range 1.45-1.99 mm) for piriform lobe and thalamus target sites, respectively. In cats, the calculated median needle placement error was 0.79 mm (range 0.6-1.91 mm) for the piriform lobe target site and 1.29 mm (range 0.47-2.69 mm) for the thalamic target site. The diagnostic yield was 96.4% (95% CI 0.81-0.99), the diagnostic accuracy was 94.4% (95% CI 0.72-0.99). Median total procedural duration for post mortem biopsies was 57.5 min (range 41-69 min). Median total procedural duration for intra vitam biopsies was 122.5 min (range 103-136 min). Three dogs were discharged 1 day after biopsy and one dog after 6 days. Two dogs and one cat were euthanized 24 and 48 h after biopsy. Intraoperative CBCT-based automated image registration for frameless stereotactic biopsies in companion animals is capable of providing diagnostic brain biopsy specimens independent of skull size and morphology with diagnostic yield and accuracy comparable to published values for diverse frameless and frame-based stereotaxy systems used in veterinary medicine. Duration of the procedure is not negatively affected and within the published range with other systems. Mobile intraoperative CBCT-based registration combined with neuronavigation delivers diagnostic brain biopsies in companion animals

Accuracy of end-on fluoroscopy in predicting implant position in relation to the vertebral canal in dogs.

Objective To evaluate the accuracy of end-on fluoroscopy in predicting implant position in relation to the vertebral canal in the canine thoracolumbar vertebral column. Study design In vitro imaging and anatomic study. Animals Canine cadaveric thoracolumbar vertebral columns (n = 5). Methods Smooth Steinmann pins were inserted bicortically into the thoracolumbar vertebral columns between T10 and L7 using recommended insertion angles. Penetration of the spinal canal was not strictly avoided. After pin placement, end-on fluoroscopy images were obtained of each pin. Pin position was subsequently assessed by four evaluators and determined to either being out of the vertebral canal or in, with the latter being additionally divided into partially or completely penetrating the canal. To assess potential differences in modalities, fluoroscopy images were gray-scale inverted and evaluated again later by the same four individuals. Correct identification of pin position in relationship to the vertebral canal was assessed for both fluoroscopy images. Anatomic preparation of the spines was used for verification of pin position in relation to the spinal canal. Some data from this study were compared with historical data on accuracy using orthogonal radiography and computed tomography (CT). Results Overall sensitivity and specificity of F to detect vertebral canal penetration was 98.8 % (95% confidence interval (CI), 96.0-99.6) and 98.0% (95% CI, 77.0-99.9), respectively. For Fi, sensitivity and specificity were 97.0% (95% CI, 91.5-99.0) and 98.5% (95% CI, 81.5-99.9) respectively. F exceeded Fi for the sensitivity of detecting pin penetration into the vertebral canal (p = 0.039) but specificities were not different (p = 0.585). When comparing to historical data, the overall accuracy of end-on fluoroscopy (F) and inverted fluoroscopy (Fi) was statistical better than conventional radiographic assessment (p < 0.001). Conclusion End-on fluoroscopy is a highly accurate method for the assessment of pin position in relationship to the thoracolumbar spinal canal in cadaveric dogs. Clinical significance End-on fluoroscopy, with or without inversion, is accurate in identifying vertebral canal violation by bicortically placed Steinmann pins. When CT is not available, end-on fluoroscopy might be a valuable imaging modality to determine pin position in the canine vertebral column

Assessment of oligoclonal bands in cerebrospinal fluid and serum of dogs with meningoencephalitis of unknown origin

Background: Meningoencephalitis of unknown origin (MUO) is an inflammatory disease of the canine central nervous system (CNS) that shares several features with multiple sclerosis (MS) in humans. In approximately 95% of MS patients, ≥ two immunoglobulin G (IgG) oligoclonal bands (OCBs) are detectable exclusively in the cerebrospinal fluid (CSF). Hypothesis/objectives: To investigate OCBs in CSF and serum in dogs affected by MUO, intervertebral disc disease (IVDD), idiopathic epilepsy (IE), intracranial neoplasia (IN), steroid-responsive meningitis-arteritis (SRMA), and diseases outside the CNS. We hypothesize that the highest prevalence of CSF-specific OCBs (≥ two OCBs uniquely in the CSF) would be found in dogs affected by MUO. Animals: Client-owned dogs (n = 121) presented to the neurology service due to neurological deficits. Methods: Prospective study. Measurement of IgG concentration in CSF and serum via a canine IgG ELISA kit. OCB detection via isoelectric focusing (IEF) and immunoblot. Results: Presence of CSF-specific OCBs was significantly higher in dogs with MUO (57%) compared to 22% in IN, 6% in IE, 15% in SRMA, 13% in IVDD, and 0% in the non-CNS group (p < .001). Dogs with MUO were 9.9 times more likely to show CSF-specific OCBs than all other diseases together (95% confidence interval, 3.7–26.4; p < .001). Conclusions and clinical importance: MUO showed the highest prevalence of CSF-specific OCBs, indicating an inflammatory B cell response. Future studies are needed to evaluate the prevalence in the specific MUO subtypes and a possible similarity with human MS

Assessment of oligoclonal bands in cerebrospinal fluid and serum of dogs with meningoencephalitis of unknown origin.

BACKGROUND Meningoencephalitis of unknown origin (MUO) is an inflammatory disease of the canine central nervous system (CNS) that shares several features with multiple sclerosis (MS) in humans. In approximately 95% of MS patients, ≥ two immunoglobulin G (IgG) oligoclonal bands (OCBs) are detectable exclusively in the cerebrospinal fluid (CSF). HYPOTHESIS/OBJECTIVES To investigate OCBs in CSF and serum in dogs affected by MUO, intervertebral disc disease (IVDD), idiopathic epilepsy (IE), intracranial neoplasia (IN), steroid-responsive meningitis-arteritis (SRMA), and diseases outside the CNS. We hypothesize that the highest prevalence of CSF-specific OCBs (≥ two OCBs uniquely in the CSF) would be found in dogs affected by MUO. ANIMALS Client-owned dogs (n = 121) presented to the neurology service due to neurological deficits. METHODS Prospective study. Measurement of IgG concentration in CSF and serum via a canine IgG ELISA kit. OCB detection via isoelectric focusing (IEF) and immunoblot. RESULTS Presence of CSF-specific OCBs was significantly higher in dogs with MUO (57%) compared to 22% in IN, 6% in IE, 15% in SRMA, 13% in IVDD, and 0% in the non-CNS group (p < .001). Dogs with MUO were 9.9 times more likely to show CSF-specific OCBs than all other diseases together (95% confidence interval, 3.7-26.4; p < .001). CONCLUSIONS AND CLINICAL IMPORTANCE MUO showed the highest prevalence of CSF-specific OCBs, indicating an inflammatory B cell response. Future studies are needed to evaluate the prevalence in the specific MUO subtypes and a possible similarity with human MS

Clinical and magnetic resonance imaging features of lymphoma involving the nervous system in cats

Background: Lymphoma is the most common spinal cord neoplasm and second most common intracranial tumor in cats, but description of specific magnetic resonance imaging (MRI) features is lacking. Objective: Describe the clinical and MRI features of lymphoma affecting the central (CNS) or peripheral (PNS) nervous system or both in cats. Animals: Thirty‐one cats with confirmed cytological or histopathological diagnosis or both of lymphoma involving the CNS or PNS or both, and MRI findings of the lesions. Methods: Multicenter retrospective descriptive study. Signalment and medical information were recorded. Magnetic resonance imaging findings were reviewed by 3 observers following a list of predefined criteria and consensus was sought. Frequency distributions of the different categorical data were reported. Results: Median duration of clinical signs at time of presentation was 14 days (range, 1‐90). Neurological examination was abnormal in 30/31 cats. On MRI, lesions affecting the CNS were diagnosed in 18/31 cats, lesions in both CNS and PNS in 12/31, and lesions in the PNS only in 1/31. Intracranial lesions were diagnosed in 22 cats (extra‐axial, 7/22; intra‐axial, 2/22; mixed, 13/22), and spinal lesions were diagnosed in 12 (6/12 involving the conus medullaris and lumbosacral plexuses). Infiltration of adjacent extra‐neural tissue was present in 11/31 cases. Contrast enhancement was seen in all lesions, being marked in 25/30. Meningeal enhancement was present in all but 2 cases. Several distinct MRI patterns were observed. Conclusions and Clinical Importance: Nervous system lymphoma in cats has a wide range of MRI features, of which none is pathognomonic. However, together with clinical data and cerebrospinal fluid (CSF) analysis, MRI may provide a strong tentative antemortem diagnosis

Comparison of nanoparticle-mediated transfection methods for DNA expression plasmids: efficiency and cytotoxicity

<p>Abstract</p> <p>Background</p> <p>Reproducibly high transfection rates with low methodology-induced cytotoxic side effects are essential to attain the required effect on targeted cells when exogenous DNA is transfected. Different approaches and modifications such as the use of nanoparticles (NPs) are being evaluated to increase transfection efficiencies. Several studies have focused on the attained transfection efficiency after NP-mediated approaches. However, data comparing toxicity of these novel approaches with conventional methods is still rare.</p> <p>Transfection efficiency and methodology-induced cytotoxicity were analysed after transfection with different NP-mediated and conventional approaches. Two eukaryotic DNA-expression-plasmids were used to transfect the mammalian cell line MTH53A applying six different transfection protocols: conventional transfection reagent (FuGENE HD, FHD), FHD in combination with two different sizes of stabilizer-free laser-generated AuNPs (PLAL-AuNPs_S1,_S2), FHD and commercially available AuNPs (Plano-AuNP), and two magnetic transfection protocols. 24 h post transfection efficiency of each protocol was analysed using fluorescence microscopy and GFP-based flow cytometry. Toxicity was assessed measuring cell proliferation and percentage of propidium iodide (PI%) positive cells. Expression of the respective recombinant proteins was evaluated by immunofluorescence.</p> <p>Results</p> <p>The addition of AuNPs to the transfection protocols significantly increased transfection efficiency in the pIRES-hrGFPII-<it>eIL-12 </it>transfections (FHD: 16%; AuNPs mean: 28%), whereas the magnet-assisted protocols did not increase efficiency. Ligand-free PLAL-AuNPs had no significant cytotoxic effect, while the ligand-stabilized Plano-AuNPs induced a significant increase in the PI% and lower cell proliferation. For pIRES-hrGFPII-<it>rHMGB1 </it>transfections significantly higher transfection efficiency was observed with PLAL-AuNPs (FHD: 31%; PLAL-AuNPs_S1: 46%; PLAL-AuNPs_S2: 50%), while the magnet-assisted transfection led to significantly lower efficiencies than the FHD protocol. With PLAL-AuNPs_S1 and _S2 the PI% was significantly higher, yet no consistent effect of these NPs on cell proliferation was observed. The magnet-assisted protocols were least effective, but did result in the lowest cytotoxic effect.</p> <p>Conclusions</p> <p>This study demonstrated that transfection efficiency of DNA-expression-plasmids was significantly improved by the addition of AuNPs. In some combinations the respective cytotoxicity was increased depending on the type of the applied AuNPs and the transfected DNA construct. Consequently, our results indicate that for routine use of these AuNPs the specific nanoparticle formulation and DNA construct combination has to be considered.</p

Otitis media and interna with or without polyps in cats:association between meningeal enhancement on postcontrast MRI, cerebrospinal fluid abnormalities, and clinician treatment choice and outcome

OBJECTIVES The aim of this study was to evaluate the association between meningeal enhancement (MgE) and cerebrospinal fluid (CSF) analysis results, their individual association with bacteriology results from affected ear samples and whether these test results influenced clinicians' therapeutic choice in cats with otitis media and interna (OMI). METHODS This was a multicentre retrospective study carried out over an 8-year period. Cats diagnosed with OMI, with or without a nasopharyngeal polyp, leading to peripheral vestibular signs were included. Only cats for which MRI with postcontrast T1-weighted sequences and CSF analyses available were included. Cats with intra-axial MRI lesions or empyema were excluded. RESULTS Fifty-eight cats met the inclusion criteria. MgE was reported in 26/58 cases, of which nine had an abnormal CSF result (increased total nucleated cell count [TNCC] or total protein); 32/58 cases had no MgE, of which 10 showed abnormal CSF results. There was no association between bacteriology results (external ear canal or bulla) and MgE or abnormal CSF results. CSF abnormalities were statistically significantly more common in acute cases (n = 16/37) than in chronic cases (n = 3/21; Fischer's test P = 0.04). Prednisolone was prescribed in 10/16 cases with increased TNCC. Among the 42 cases with normal TNCC, 15 received prednisolone and 13 received non-steroidal anti-inflammatory drugs. Various antimicrobial drugs were prescribed in 53/58 cats. Duration of antimicrobial treatment was similar, regardless of positive bacterial culture (5.58 vs 4.22 weeks), abnormal CSF (5.83 vs 4.76 weeks) or MgE (5.33 vs 4.90 weeks). CONCLUSIONS AND RELEVANCE No association was found between the CSF and MgE results. Furthermore, no association was found between MgE, CSF or bacteriology findings. In addition, abnormal CSF results might lead the clinician to treat with corticosteroids, but they did not have any impact on duration of antimicrobial treatment. CSF abnormalities were seen significantly less frequently in chronic cases. The outcome tended to be poorer when MgE was detected on MRI