The parents and first- to fifth-generation offspring in a single family of Shetland sheepdogs were studied (). The female parent dog produced 6 litters, for a total of 28 puppies. At the time when 3 litters had been produced, a 2-year-old female from the second litter (dog 1) suddenly showed a generalized seizure and was referred to a veterinary clinic for clinical diagnosis. She did not show any abnormalities upon examination of the urine and the blood. She showed generalized epilepsy for 3 y. She died after status epilepticus at age of 5 y, and then was submitted to our laboratory for pathologic diagnosis. Most dogs in the first 4 litters of first-, second-, and third-generation offspring were not available for evaluation, as the puppies had been given away without obtaining information about the new owner. Dogs from a litter of first-generation (litter no. 6) and fifth-generation (litter no. 17) puppies, which were produced for this study, were evaluated propectively. A conventional vaccination schedule was followed, and a monthly heartworm preventive treatment was administered. Appropriate veterinary care was provided if a generalized seizure occurred. This study was carried out under the control of the Animal Research Committee, in accordance with the guidelines for animal experimentation of the Faculty of Agriculture, Tottori University.
Figure 1. Placement of the subcutaneous electrodes for electroencephalographic recording in epileptic and control dogs. F1 and F3 — left frontal; F2 and F5 — right frontal; central frontal (F4), left parietal (P1); right parietal (P2); (more ...)
Examination and clinicopathologic evaluations
Six dogs from 2 litters (nos. 6 and 17), which were produced for this study, were evaluated every 1 to 2 mo by EEG, beginning at 8 mo of age and continuing until they developed a generalized seizure. One dog (dog 3; litter 5) from the first-generation puppies was evaluated 4 times by EEG at 6, 9, 13, and 15 mo of age. Three dogs (litter 13) from the fourth-generation puppies were evaluated 4 times by EEG, at 9, 12, 16, and 18 mo of age. One dog (dog 7; litter 15) from the fifth-generation puppies was evaluated twice by EEG, at 6 and 12 mo of age. Electoencephalographic and histopathologic examinations were performed on 6 dogs (dogs 3–7, 10). Two dogs (dogs 1 and 2) were examined by histopathology only, and 4 other dogs from 2 litters (litters 6 and 17) were analyzed only by EEG.
Hematologic examination was performed on all the dogs mentioned above. Complete blood counts were determined by using standard methods. Serum electrolyte and biochemical analyses (albumin, creatine phosphokinase, blood urea nitrogen, cholesterol, total calcium, chloride, sodium, potassium, inorganic phosphorus, glucose, and total protein) were performed by using 2 autobiochemical analyzers (Dry-Chem 800 and Dry-Chem 3500I; Fyji Photo Film, Tokyo, Japan).
Electroencephalographic examination was performed every 1 to 3 mo. The EEG was recorded when the dogs were under sedation by IM injection of xylazine (Celactal; Bayer, Leverkussen, Germany), 1.0 mg/kg body weight (BW). Before recording the EEG, each dog was placed in a dimly lighted, sound-attenuated room. Four female age-matched Shetland sheepdogs without seizure history, from 1 to 2 y of age, were used as control cases.
All the dogs were submitted to a single recording using a standard 10-20 surface electrodes, as used in humans (14
) (). Before insertion of the electrodes, local subcutaneous infiltration anesthesia with 1% lidocaine hydrochloride (Xylocaine; Fujisawa Pharmaceuticals, Tokyo, Japan) was administered around the placement sites. The electrodes (stainless-steel needles) were inserted subcutaneously, bilaterally over the left frontal, right frontal, central frontal, left parietal, right parietal, central parietal, left temporal, right temporal , left occipital, right occipital, and vertex areas. Reference and ground electrodes were also inserted just behind the tip of the nose and at the caudal edge of the external occipital protuberance, respectively. Connections to the recording devices were made via flexible cable. A 12-channel electroencephalograph was used for recording the EEG, using a polygraph (No. 1A52; NEC San-ei, Tokyo, Japan) with a time constant of 0.3 s, amplification sensitivity of 50 μV/5 mm, and high-frequency filter of 60 Hz (16
). The EEG signals were recorded on paper for visual analysis (paper speed, 3 cm/s).
Cerebrospinal fluid collection
Cerebomedullary cisternal cerebrospinal fluid (CSF) was collected from 6 epileptic dogs that were clinically normal, from 1 dog that had died after status epilepticus, and from 4 female, age-matched control Shetland sheepdogs. In all epileptic dogs, collection of CSF was done at 1 wk (or longer) after the last observed seizure. All dogs were anesthetized by IM injections of medetomidine hydrochloride (Domitor; Meiji Seika, Tokyo, Japan), 30 μg/kg BW; midazolam (Dormicum; Yamanouchi Pharmaceutical, Tokyo, Japan), 0.2 mg/kg BW; and ketamine hydrochloride (Ketalal; Sankyo Pharmaceutical, Tokyo, Japan), 5.0 mg/kg BW. After induction of anesthesia, the dogs were intubated and the hair over a 4-cm × 6-cm area, from the occipital protuberance to the mid-body of the second cervical vertebra, was shaved, and the skin was prepared for CSF collection. The CSF was collected aseptically by using a 22-gauge, 2-inch spinal needle and the samples were immediately frozen in liquid nitrogen. The samples were then transferred to a refrigerator where they were kept at −80°C until analysis.
Cerebrospinal fluid from all dogs was analyzed for levels of aspartate, glutamate, asparagine, glutamine, arginine, taurine, and gamma-aminobutyric acid (GABA). Continuous sample analysis was done by high performance liquid chromatography with electrochemical detection (HPLC-ECD) (BAA-300; Eicom, Kyoto, Japan). Samples from epileptic and control dogs were randomly interspersed during analysis.
Necropsy and histopathology
Complete postmortem examinations were performed on 7 affected dogs. Systemic organs were fixed by immersion in 10% neutral buffered formalin, routinely processed into paraffin, and sectioned at 4 μm. Sections were stained with hematoxylin and eosin, combined luxol-fast-blue-hematoxylin-eosin, Klüver-Barrera, modified Bielschowsky, and Gallyas-Braak stains.
Immunohistochemical analysis was performed using an anti-human glial fibrillary acid protein (GFAP) mouse monoclonal antiserum (DAKO, Glostrup, Denmark) in an indirect immunoperoxidase staining procedure. Briefly, after blocking endogeneous peroxidase activity with 3% hydrogen peroxide, sections were preincubated with 10% normal goat serum for 30 min at room temperature, incubated with the primary antibody (1:100 dilution) at 4°C overnight, and then incubated with biotinylated goat anti-mouse IgG antiserum (1:400; DAKO) at room temperature, followed by peroxidase-conjugated streptavidin (1:400; DAKO) for 30 min. After the first and second incubations, the sections were given 5-minute washes in phosphate-buffered saline (PBS), and the immunoreaction was developed in substrate solution containing 0.05% 3,3-diaminobenzidine tetrahydrochloride and 0.003% hydrogen peroxide. Sections were counterstained lightly with hematoxylin. Specificity controls were carried out by replacing the primary antibody with PBS. Samples from 2 beagles, 3 mongrels, and 2 Shetland sheepdogs that had died of non-neurological disorders served as controls.