Holger A. Volk

Epileptogenesis and neuropathology after different types of status epilepticus induced by prolonged electrical stimulation of the basolateral amygdala in rats

It has previously been shown that prolonged (60-min) low-intensity electrical stimulation of a kindled focus in the basolateral nucleus of the amygdala (BLA) of Wistar rats resulted in the development of self-sustained status epilepticus (SSSE) with predominantly partial seizures and subsequent brain damage in the ipsilateral hemisphere. In the present study, using high-intensity (700 microA) pulsed-train electrical stimulation of the BLA for 25 min, SSSE was induced in both kindled and non-kindled Wistar rats, demonstrating that under these experimental conditions prior kindling is not necessary to induce SSSE. Thus, all subsequent experiments were done in non-kindled rats of different strains (Wistar, Sprague-Dawley) and genders. Three distinct behavioral types of SSSE were observed: (1) continuous partial seizures; (2) continuous partial seizures, repeatedly interrupted by generalized convulsive seizures; and (3) continuous generalized convulsive seizures. These three forms of SSSE were seen in both strains and genders, although the percentage of rats in each strain and gender developing a specific type of SSSE differed. Rats spontaneously recovered from SSSE after between 3 and 8h on average, the SSSE duration depending on SSSE type, rat strain and gender. Following SSSE, rats were monitored with a video- and EEG-recording system for occurrence of spontaneous recurrent seizures (SRS). Overall, about 80% of the rats developed epilepsy with SRS after SSSE, but the proportion of rats developing SRS depended on the type of SSSE. Only 33% of the rats developed SRS after a partial SSSE, compared to >90% in case of either type 2 or type 3 SSSE with generalized convulsive seizures. Interruption of different forms of SSSE with diazepam after 90 min prevented development of epilepsy, while a generalized SSSE duration of 4h consistently produced epilepsy in >90% of rats. Histologic analysis of rat brains after the different SSSE types indicated that neuronal loss after partial SSSE was much more regionally restricted and less severe compared to neuronal damage after SSSE with generalized convulsive seizures, which was similar to the brain damage seen in the kainate and pilocarpine models of temporal lobe epilepsy. These experiments establish that prolonged electrical stimulation of the BLA induces different forms of SSSE that resemble nonconvulsive and convulsive types of SE in humans. These different forms of SSSE induce epilepsy with SRS and brain pathology reminiscent of temporal lobe epilepsy with hippocampal sclerosis. The rat model provides a new tool to mimic different types of SE and investigate the pathogenesis underlying their long-term complications.

Immunohistochemical Localization of P-glycoprotein in Rat Brain and Detection of Its Increased Expression by Seizures Are Sensitive to Fixation and Staining Variables

The MDR1 gene product, P-glycoprotein (P-gp), was shown to confer multi-drug resistance to cancer cells, but its overexpression is also suggested to be involved in pharmacoresistance of epilepsy by acting as an energy-dependent drug-efflux pump in the blood-brain barrier (BBB). In normal brain tissue, P-gp is almost exclusively expressed by capillary endothelial cells (EC) of the BBB, whereas little or no expression is detected in other cell types. Increased P-gp expression was observed after seizures, but localization of this increase, i.e., within brain capillary EC or within parenchymal or perivascular astrocytes, which contribute to the BBB function, is controversial. To test whether these antithetic data arise from unusual properties of the antigen itself, we compared different immunohistochemical techniques and monoclonal or polyclonal antibodies to P-gp in normal rat brain and rat brain after kainate-induced seizures. Using acetone-fixed cryostat sections of snap-frozen tissue, strong P-gp labeling was detected in EC and, after seizures, in hippocampal neurons, but not in astrocytes. In contrast, EC and neuronal P-gp immunolabeling were not seen in paraformaldehyde-fixed sections, whereas both perivascular and parenchymal astrocytes exhibited strong P-gp labeling after seizures. The lack of P-gp labeling in EC by paraformaldehyde fixation, was reversed by treatment of the sections with acetate/ethanol. These experiments demonstrate that various fixation conditions have a striking effect on the immunohistochemical localization of P-gp in rat brain and detection of its increased expression by seizures. When data obtained from different immunohistochemical techniques are taken together, seizures seem to induce overexpression of P-gp in four different cell types, i.e., EC, perivascular astrocytes, parenchymal astrocytes, and neurons.

Increased expression of the multidrug transporter P-glycoprotein in limbic brain regions after amygdala-kindled seizures in rats

Increased expression of the multidrug transporter P-glycoprotein (Pgp; ABCB1) has previously been found in epileptogenic brain tissue from patients with pharmacoresistant temporal lobe epilepsy (TLE) as well as in the hippocampus and other limbic brain regions in the rat kainate model of TLE. Approaches to the quantification of Pgp expression have mainly been based on subjective visual estimation of the level of Pgp immunoreactivity in brain sections. In the present study, computer-assisted image analysis based on optical density (OD) measurements was used to examine immunohistochemical expression of Pgp in the kindling model of TLE. Sections from kainate-treated rats were used for comparison. Using diaminobenzidine as chromogen, Pgp was exclusively located in brain capillary endothelial cells, which was confirmed by double-labeling with an antibody against the endothelial glucose transporter (GLUT-1). After kainate-induced seizures, the intensity of endothelial Pgp staining significantly increased by 70-80% in the dentate gyrus. A significant, albeit less marked increase in Pgp expression in this area was also seen after amygdala-kindled seizures. Furthermore, Pgp was upregulated after kindling in the hilus of the dentate gyrus, the CA1 and CA3 sectors of the hippocampus, and the piriform and cerebral cortex. In kindled rats, most Pgp alterations occurred ipsilateral to the electrode in the basolateral amygdala. The data demonstrate that computer-assisted image analysis using OD is an accurate and rapid method to determine the relative amount of Pgp protein in brain sections and the effects of seizures on this multidrug transporter. The fact that Pgp overexpression in brain capillary endothelial cells occurs in two established models of difficult-to-treat TLE substantiates the notion that seizure-induced upregulation of Pgp contributes to multidrug resistance (MDR) in epilepsy.

Prevalence and risk factors for canine epilepsy of unknown origin in the UK

Epidemiological evaluation of canine epilepsy is an under-researched area. The objectives of this study were to estimate prevalence and investigate risk factors for epilepsy of unknown origin (EUO) among dogs attending primary veterinary practices in the UK. The clinical data analysed spanned a two-year period and included all dogs attending 92 primary veterinary clinics participating in the VetCompass project. Five hundred and thirty-nine EUO cases were identified giving a prevalence of 0.62% (95% CI 0.57% to 0.67%). Males were over 1.5 times as likely to have EUO compared with females (95% CI 1.44 to 2.06; P < 0.001). Of purebred dogs, the border terrier had 2.70 (95% CI 1.57 to 4.62; P < 0.001) and the German shepherd dog had 1.90 (95% CI 1.28 to 2.80; P=0.001) times increased odds of EUO compared with crossbred dogs. In addition, the West Highland white terrier had reduced odds (OR 0.23; 95% CI 0.08 to 0.62; P=0.004) of EUO compared with crossbred dogs (likelihood ratio test P < 0.0001). No association was found with neuter status, colour or weight. The current study highlights the clinical importance of epilepsy as a canine disorder in the UK. Increased awareness of sex and breed predispositions may assist clinicians with diagnosis. Further research is merited to evaluate the specific breed associations identified.

Striking differences in individual anticonvulsant response to phenobarbital in rats with spontaneous seizures after status epilepticus.

Summary:  Purpose: More than one third of patients with epilepsy have inadequate control of seizures with drug therapy, but mechanisms of intractability are largely unknown. Because of this large number of pharmacoresistant patients with epilepsy, the existing process of antiepileptic drug (AED) discovery and development must be reevaluated with a focus on preclinical models of therapy‐resistant epilepsy syndromes such as mesial temporal lobe epilepsy (TLE). However, although various rodent models of TLE are available, the pharmacoresponsiveness of most models is not well known. In the present study, we used a post–status epilepticus model of TLE to examine whether rats with spontaneous recurrent seizures (SRSs) differ in their individual responses to phenobarbital (PB).

International veterinary epilepsy task force consensus report on epilepsy definition, classification and terminology in companion animals

Dogs with epilepsy are among the commonest neurological patients in veterinary practice and therefore have historically attracted much attention with regard to definitions, clinical approach and management. A number of classification proposals for canine epilepsy have been published during the years reflecting always in parts the current proposals coming from the human epilepsy organisation the International League Against Epilepsy (ILAE). It has however not been possible to gain agreed consensus, “a common language”, for the classification and terminology used between veterinary and human neurologists and neuroscientists, practitioners, neuropharmacologists and neuropathologists. This has led to an unfortunate situation where different veterinary publications and textbook chapters on epilepsy merely reflect individual author preferences with respect to terminology, which can be confusing to the readers and influence the definition and diagnosis of epilepsy in first line practice and research studies.In this document the International Veterinary Epilepsy Task Force (IVETF) discusses current understanding of canine epilepsy and presents our 2015 proposal for terminology and classification of epilepsy and epileptic seizures. We propose a classification system which reflects new thoughts from the human ILAE but also roots in former well accepted terminology. We think that this classification system can be used by all stakeholders.

Prophylactic treatment with levetiracetam after status epilepticus: lack of effect on epileptogenesis, neuronal damage, and behavioral alterations in rats.

Levetiracetam (LEV) is a structurally novel antiepileptic drug (AED) which has demonstrated a broad spectrum of anticonvulsant activities both in experimental and clinical studies. Previous experiments in the kindling model suggested that LEV, in addition to its seizure-suppressing activity, may possess antiepileptogenic or disease-modifying activity. In the present study, we evaluated this possibility by using a rat model in which epilepsy with spontaneous recurrent seizures (SRS), behavioral alterations, and hippocampal damages develop after a status epilepticus (SE) induced by sustained electrical stimulation of the basal amygdala. Two experimental protocols were used. In the first protocol, LEV treatment was started 24h after onset of electrical amygdala stimulation without prior termination of the SE. In the second protocol, the SE was interrupted after 4h by diazepam, immediately followed by onset of treatment with LEV. Treatment with LEV was continued for 8 weeks (experiment #1) or 5 weeks (experiment #2) after SE, using continuous drug administration via osmotic minipumps. The occurrence of SRS was recorded during and after treatment. In addition, the rats were tested in a battery of behavioral tests, including the elevated-plus maze and the Morris water maze. Finally, the brains of the animals were analyzed for histological lesions in the hippocampal formation. With the experimental protocols chosen for these experiments, LEV did not exert antiepileptogenic or neuroprotective activity. Furthermore, the behavioral alterations, e.g., behavioral hyperexcitability and learning deficits, in epileptic rats were not affected by treatment with LEV after SE. These data do not support the idea that administration of LEV after SE prevents or reduces the long-term alterations developing after such brain insult in rats.

Antiepileptic drug-resistant rats differ from drug-responsive rats in hippocampal neurodegeneration and GABAA receptor ligand binding in a model of temporal lobe epilepsy

The disabling seizures associated with mesial temporal lobe epilepsy (TLE) are often resistant to antiepileptic drugs (AEDs). The biological basis of this refractoriness is unknown but may include alterations in AED targets in the epileptogenic brain tissue, reduced AED penetration to the seizure focus, and neuropathological brain alterations such as hippocampal sclerosis typically found in patients with refractory TLE. In the present study, we used a rat model of TLE to examine whether AED responders differ from non-responders in their structural alterations and GABA(A) receptor characteristics in the hippocampal formation. In this model, spontaneous recurrent seizures develop after a status epilepticus induced by prolonged electrical stimulation of the basolateral amygdala. The frequency of these seizures was recorded by continuous video/EEG monitoring before, during, and after daily treatment with phenobarbital, which was given at maximum tolerated doses for 2 weeks. Based on their individual response to phenobarbital, rats were grouped into responders and non-responders. The severity or duration of the initial brain insult (the status epilepticus) did not differ between responders and non-responders, indicating that the difference between the two subgroups is genetically determined. Subsequent histological examination showed a significant loss of neurons in the CA1, CA3c/CA4, and dentate hilus of non-responders, whereas responders did not differ in this respect from non-epileptic controls. The morphological alterations in the non-responders were associated with striking alterations in autoradiographic imaging of diazepam-sensitive and diazepam-insensitive GABA(A) receptor binding in the dentate gyrus with a significant shift to enhanced diazepam-insensitive binding. The present data indicate that neurodegeneration and associated GABA(A) receptor changes in the dentate gyrus are critically involved in the mechanisms underlying refractoriness of seizures in TLE.

Expression of the multidrug transporter MRP2 in the blood-brain barrier after pilocarpine-induced seizures in rats

Multidrug resistance proteins (MRPs; symbol ABCC) are membrane glycoproteins that mediate the ATP-dependent export of a wide range of substrates from cells and thereby affect the bioavailability and disposition of many drugs. MRP2 (ABCC2) is expressed on the apical domain of hepatocytes, enterocytes of the proximal small intestine, and proximal renal tubular cells, but its location in the brain is a matter of debate. Most previous studies failed to determine MRP2 mRNA or protein in the brain or cell preparations from the brain of different species including humans. Based on our previous experience with the drug efflux transporter P-glycoprotein, we evaluated whether the immunohistochemical determination of MRP2 expression is sensitive to fixation and staining variables. Furthermore, we examined whether the MRP2 protein is overexpressed after experimentally induced seizures in rats, using the pilocarpine model of temporal lobe epilepsy. The MRP2 expression in the liver was used as positive control. MRP2 deficient TR- rats were used as negative controls. Despite various modifications in tissue fixation and immunohistochemical staining as well as use of different commercially available MRP2 antibodies, we never observed any unequivocal MRP2 staining in the brain of normal rats. However, after a pilocarpine-induced convulsive status epilepticus, clear MRP2 staining became visible in brain capillary endothelial cells and, less frequently, perivascular astroglia and neurons in various brain regions. In view of our recent data on brain access of antiepileptic drugs in MRP2 deficient TR- rats, seizure-induced over-expression of MRP2 in the blood-brain barrier is likely to impair drug penetration into the brain, thereby contributing to drug resistance in epilepsy.

Relationship of brain parenchyma within the caudal cranial fossa and ventricle size to syringomyelia in cavalier King Charles spaniels.

OBJECTIVES To assess if the volumes of the caudal cranial fossa (CCF), parenchyma within the caudal cranial fossa (CCFP) or ventricles (V) are associated with syringomyelia (SM) in cavalier King Charles spaniels (CKCS) with Chiari-like malformation (CM). To evaluate if volumes are associated with transverse syrinx width. METHODS Magnetic resonance images of 59 CKCS with CM were retrospectively reviewed and grouped with or without SM. Three-dimensional images were created and volumes of the fossae, brain parenchyma and ventricular system were calculated from which percentages of CCF, CCFP and V were created. If present, syrinx size was measured from its maximal transverse width. The percentages were statistically compared between groups, and correlation between percentages and syrinx dimensions was made. RESULTS CKCS with SM had significantly higher CCFP (P=0.0001) and V (P=0.0002) to those without but no significant difference in CCF (P=0.925). There was a positive correlation between CCFP and syrinx width (Pearson r=0.437) and ventricle size to syrinx width (Spearman r=0.627). CLINICAL SIGNIFICANCE A more marked overcrowding of the CCF is associated with SM, which may explain the high incidence of SM in CKCS with CM. The association between ventricle and syrinx dimensions supports the theory that SM development is the result of altered cerebrospinal fluid dynamics.