Diagnostic utility of cerebrospinal fluid analysis in dogs with suspected idiopathic epilepsy.

BACKGROUND: Idiopathic epilepsy (IE) is the most common cause of repeated seizures in dogs. The International Veterinary Epilepsy Task Force consensus guidelines recommend performing magnetic resonance imaging (MRI) of the brain and cerebrospinal fluid (CSF) analysis as part of a tier II diagnosis of IE, and these procedures have documented risks. The aim of this retrospective study was to identify how often dogs with suspected IE have abnormalities on CSF analysis. METHODS: Dogs aged between 6 months and 6 years that were presented with a history of two or more seizures with at least 24 h between seizure episodes, a normal neurologic examination, no evidence of toxic or metabolic causes, a normal MRI scan (including contrast administration) and CSF analysis were included. RESULTS: Eighty-two dogs were included. Of these, nine dogs (10.9%) had abnormalities on CSF analysis: five of nine dogs (55.5%) had albuminocytologic dissociation, three of nine dogs (33.3%) had mild increases in total nucleated cell count (TNCC), and one of nine dogs (11.1%) had mild increase in both total protein and TNCC. Cytology in dogs with elevated TNCC revealed a mononuclear pleocytosis. One of the nine dogs with abnormal CSF had a seizure within the 24 h before investigations, and six of nine dogs had a seizure within 1 month before investigation. CONCLUSION: CSF analysis can play an important role in the diagnostic investigation of the underlying causes of repeated seizures. However, in dogs with a normal inter-ictal neurological examination and MRI scan, it rarely reveals significant abnormalities, and the risk of performing a CSF tap may outweigh the potential diagnostic gain.

Is there a link between bacteriuria and a reversible encephalopathy in dogs and cats?

Bacteriuria has been associated with abnormal neurological status in humans, especially geriatric patients. In this report, we review 11 cases (seven dogs and four cats) that suggest an association between bacteriuria and abnormal neurological status in veterinary medicine. These cases showed diffuse forebrain signs with or without brainstem signs, but primary brain disease was excluded by MRI and cerebrospinal fluid analysis. Bacteriological culture of urine was positive in each animal and neurological deficits improved or resolved with initiation of antibiosis ± fluid therapy and levetiracetam. While further studies are needed to definitively confirm or refute the link between bacteriuria and a reversible encephalopathy, urine bacteriological culture should be considered in veterinary patients presented with acute onset forebrain neuro-anatomical localisation, even in the absence of clinical signs of lower urinary tract inflammation.

Clinical presentation and magnetic resonance imaging findings in 11 dogs with eosinophilic meningoencephalitis of unknown aetiology.

OBJECTIVES: To describe the clinical presentation, MRI findings and outcome in dogs with eosinophilic meningoencephalitis of unknown origin. MATERIALS AND METHODS: Dogs were included in this retrospective study if they had complete medical records, complete neurological examination, MR imaging, cerebellomedullary cerebrospinal fluid sample consistent with eosinophilic pleocytosis and negative infectious disease testing. RESULTS: Eleven dogs were included with a median age of 22·0 months (range 7·6 to 92·0 months). Nine breeds were represented. Neurological abnormalities included obtundation (n=10), menace response deficits (n=9), proprioceptive deficits (n=7), ataxia (n=7) and seizures (n=2). Neuroanatomical localisation was multi-focal (n=4), central vestibular system (n=4), diffuse forebrain (n=2) or left trigeminal/facial nerves (n=1). Seven dogs had peripheral eosinophilia. Ten dogs had bilateral symmetrical lesions affecting the cortical grey matter, which was hyperintense on T2-weighted and fluid-attenuating inversion recovery images and iso- to hypointense on T1-weighted images with associated meningeal contrast enhancement. MRI findings were consistent with diffuse meningitis and atrophy or necrosis of cortical grey matter. One dog had increased contrast uptake in the left trigeminal nerve. Ten dogs receiving corticosteroids survived to discharge, with seven also receiving cytarabine arabinoside. Median survival time was 762 days. CLINICAL SIGNIFICANCE: Eosinophilic meningoencephalitis of unknown origin affects younger larger-breed dogs, with the majority having suspected diffuse cerebrocortical meningitis and cortical (polio)encephalitis, which can be identified on MRI. Response to immunosuppressive treatment is good in the medium to long term, although further studies are required in this area.

Clinical reasoning in canine spinal disease: what combination of clinical information is useful?

Spinal disease in dogs is commonly encountered in veterinary practice. Numerous diseases may cause similar clinical signs and presenting histories. The study objective was to use statistical models to identify combinations of discrete parameters from the patient signalment, history and neurological examination that could suggest the most likely diagnoses with statistical significance. A retrospective study of 500 dogs referred to the Queen Mother Hospital for Animals before June 2012 for the investigation of spinal disease was performed. Details regarding signalment, history, physical and neurological examinations, neuroanatomical localisation and imaging data were obtained. Univariate analyses of variables (breed, age, weight, onset, deterioration, pain, asymmetry, neuroanatomical localisation) were performed, and variables were retained in a multivariate logistic regression model if P<0.05. Leading diagnoses were intervertebral disc extrusion (IVDE, n=149), intervertebral disc protrusion (n=149), ischaemic myelopathy (IM, n=48) and neoplasms (n=44). Multivariate logistic regression characterised IM and acute non-compressive nucleus pulposus extrusions as the only peracute onset, non-progressive, non-painful and asymmetrical T3-L3 myelopathies. IVDE was most commonly characterised as acute onset, often deteriorating, painful and largely symmetrical T3-L3 myelopathy. This study suggests that most spinal diseases cause distinctive combinations of presenting clinical parameters (signalment, onset, deterioration, pain, asymmetry, neuroanatomical localisation). Taking particular account of these parameters may aid decision making in a clinical setting.

Risk factors for early post-operative neurological deterioration in dogs undergoing a cervical dorsal laminectomy or hemilaminectomy: 100 cases (2002-2014).

Early post-operative neurological deterioration is a well-known complication following dorsal cervical laminectomies and hemilaminectomies in dogs. This study aimed to evaluate potential risk factors for early post-operative neurological deterioration following these surgical procedures. Medical records of 100 dogs that had undergone a cervical dorsal laminectomy or hemilaminectomy between 2002 and 2014 were assessed retrospectively. Assessed variables included signalment, bodyweight, duration of clinical signs, neurological status before surgery, diagnosis, surgical site, type and extent of surgery and duration of procedure. Outcome measures were neurological status immediately following surgery and duration of hospitalisation. Univariate statistical analysis was performed to identify variables to be included in a multivariate model. Diagnoses included osseous associated cervical spondylomyelopathy (OACSM; n = 41), acute intervertebral disk extrusion (IVDE; 31), meningioma (11), spinal arachnoid diverticulum (10) and vertebral arch anomalies (7). Overall 54% (95% CI 45.25-64.75) of dogs were neurologically worse 48 h post-operatively. Multivariate statistical analysis identified four factors significantly related to early post-operative neurological outcome. Diagnoses of OACSM or meningioma were considered the strongest variables to predict early post-operative neurological deterioration, followed by higher (more severely affected) neurological grade before surgery and longer surgery time. This information can aid in the management of expectations of clinical staff and owners with dogs undergoing these surgical procedures.

Interactions between inositol trisphosphate receptors and fluorescent Ca2+ indicators.

Fura-2 and BAPTA were previously shown to be competitive antagonists of inositol trisphosphate (InsP3) receptors, but for practical reasons the analyses were performed at pH 8.3. We recently developed a scintillation proximity assay (SPA) for pure cerebellar InsP3 receptors which allows low affinity interactions to be characterized and is readily applicable to scarce or expensive ligands. In the present study, we use SPA to demonstrate that at pH 7.2, many of the commonly used fluorescent Ca2+ indicators reversibly displace 3H-InsP3 from its receptor and that they differ substantially in their affinities for the InsP3 receptor (IC50 = 6.5-137 microM). Recombinant type 1 InsP3 receptors expressed in Sf9 cells were used to examine 3H-InsP3 binding in cytosol-like medium: both fura-2 (IC50 = 796 +/- 86 microM) and Ca Green-5N (IC50 = 62 +/- 7 microM) completely inhibited the binding, but only in their Ca(2+)-free forms. Similar results were obtained with type 3 InsP3 receptors. We conclude that many Ca2+ indicators in their Ca(2+)-free forms compete with InsP3 for binding to its receptor, and that for Ca Green-5N the interaction occurs with sufficient affinity to significantly perturb physiological responses.

A novel role for calmodulin: Ca2+-independent inhibition of type-1 inositol trisphosphate receptors.

Calmodulin inhibits both inositol 1,4,5-trisphosphate (IP3) binding to, and IP3-evoked Ca2+ release by, cerebellar IP3 receptors [Patel, Morris, Adkins, O'Beirne and Taylor (1997) Proc. Natl. Acad. Sci. U. S.A. 94, 11627-11632]. In the present study, full-length rat type-1 and -3 IP3 receptors were expressed at high levels in insect Spodoptera frugiperda 9 cells and the effects of calmodulin were examined. In the absence of Ca2+, calmodulin caused a concentration-dependent and reversible inhibition of [3H]IP3 binding to type-1 IP3 receptors by decreasing their apparent affinity for IP3. The effect was not reproduced by high concentrations of troponin C, parvalbumin or S-100. Increasing the medium free [Ca2+] ([Ca2+]m) inhibited [3H]IP3 binding to type-1 receptors, but the further inhibition caused by a submaximal concentration of calmodulin was similar at each [Ca2+]m. In the absence of Ca2+, 125I-calmodulin bound to a single site on each type-1 receptor subunit and to an additional site in the presence of Ca2+. There was no detectable binding of 125I-calmodulin to type-3 receptors and binding of [3H]IP3 was insensitive to calmodulin at all [Ca2+]m. Both peptide and conventional Ca2+-calmodulin antagonists affected neither [3H]IP3 binding directly nor the inhibitory effect of calmodulin in the absence of Ca2+, but each caused a [Ca2+]m-dependent reversal of the inhibition of [3H]IP3 binding caused by calmodulin. Camstatin, a peptide that binds to calmodulin equally well in the presence or absence of Ca2+, reversed the inhibitory effects of calmodulin on [3H]IP3 binding at all [Ca2+]m. We conclude that calmodulin specifically inhibits [3H]IP3 binding to type-1 IP3 receptors: the first example of a protein regulated by calmodulin in an entirely Ca2+-independent manner. Inhibition of type-1 IP3 receptors by calmodulin may dynamically regulate their sensitivity to IP3 in response to the changes in cytosolic free calmodulin concentration thought to accompany stimulation of neurones.

Differential regulation of types-1 and -3 inositol trisphosphate receptors by cytosolic Ca2+.

Biphasic regulation of inositol trisphosphate (IP3)-stimulated Ca2+ mobilization by cytosolic Ca2+ is believed to contribute to regenerative intracellular Ca2+ signals. Since cells typically express several IP3 receptor isoforms and the effects of cytosolic Ca2+ are not mediated by a single mechanism, it is important to resolve the properties of each receptor subtype. Full-length rat types-1 and -3 IP3 receptors were expressed in insect Sf9 cells at levels 10-40-fold higher than the endogenous receptors. The expressed receptors were glycosylated and assembled into tetramers, and binding of [3H]IP3 to each subtype was regulated by cytosolic Ca2+. The effects of increased [Ca2+] on native cerebellar and type-1 receptors expressed in Sf9 cells were indistinguishable. A maximally effective increase in [Ca2+] reversibly inhibited [3H]IP3 binding by approx. 50% by decreasing the number of IP3-binding sites (Bmax) without affecting their affinity for IP3. The effects of Ca2+ on type-3 receptors were more complex: increasing [Ca2+] first stimulated [3H]IP3 binding by increasing Bmax, and then inhibited it by causing a substantial decrease in the affinity of the receptor for IP3. The different effects of Ca2+ on the receptor subtypes were not a consequence of limitations in the availability of accessory proteins or of artifactual effects of Ca2+ on membrane structure. We conclude that Ca2+ can inhibit IP3 binding to types-1 and -3 IP3 receptors although by different mechanisms, and that IP3 binding to type-3 receptors is stimulated at intermediate [Ca2+]. A consequence of these differences is that, at resting cytosolic [Ca2+], type-3 receptors are more sensitive than type-1 receptors to IP3, but the situation reverses at higher cytosolic [Ca2+]. Such differences may be important in generating the spatially and temporally complex changes in cytosolic [Ca2+] evoked by receptors linked to IP3 formation.

Type 3 inositol trisphosphate receptors in RINm5F cells are biphasically regulated by cytosolic Ca2+ and mediate quantal Ca2+ mobilization.

There are three subtypes of mammalian Ins(1,4,5)P(3) (InsP(3)) receptor, each of which forms an intracellular Ca(2+) channel. Biphasic regulation of InsP(3) receptors by cytosolic Ca(2+) is well documented in cells expressing predominantly type 1 or type 2 InsP(3) receptors and might contribute to the regenerative recruitment of Ca(2+) release events and to limiting their duration in intact cells. The properties of type 3 receptors are less clear. Bilayer recording from InsP(3) receptors of RIN-5F cells, cells in which the InsP(3) receptors are likely to be largely type 3, recently suggested that the receptors are not inhibited by Ca(2+) [Hagar, Burgstahler, Nathanson and Ehrlich (1998) Nature (London) 296, 81-84]. By using antipeptide antisera that either selectively recognized each InsP(3) receptor subtype or interacted equally well with all subtypes, together with membranes from Spodoptera frugiperda (Sf9) cells expressing only single receptor subtypes to calibrate the immunoblotting, we quantified the relative levels of expression of type 1 (17%) and type 3 (77%) InsP(3) receptors in RINm5F cells. In unidirectional (45)Ca(2+) efflux experiments from permeabilized RINm5F cells, submaximal concentrations of InsP(3) released only a fraction of the InsP(3)-sensitive Ca(2+) stores, indicating that responses to InsP(3) are quantal. Increasing the cytosolic free [Ca(2+)] ([Ca(2+)](i)) from approx. 4 to 186 nM increased the sensitivity of the Ca(2+) stores to InsP(3): the EC(50) decreased from 281+/-15 to 82+/-2 nM. Further increases in [Ca(2+)](i) massively decreased the sensitivity of the stores to InsP(3), by almost 10-fold when [Ca(2+)](i) was 2.4 microM, and by more than 3000-fold when it was 100 microM. The inhibition caused by 100 microM Ca(2+) was fully reversed within 60 s of the restoration of [Ca(2+)](i) to 186 nM. The effect of submaximal InsP(3) concentrations on Ca(2+) mobilization from permeabilized RINm5F cells is therefore biphasically regulated by cytosolic Ca(2+). We conclude that type 3 InsP(3) receptors of RINm5F cells mediate quantal Ca(2+) release and they are biphasically regulated by cytosolic Ca(2+), either because a single type 1 subunit within the tetrameric receptor confers the Ca(2+) inhibition or because the type 3 subtype is itself directly inhibited by Ca(2+).