Neuropathologic features of the hippocampus and amygdala in cats with familial spontaneous epilepsy

Yoshihiko Yu Department of Clinical Veterinary Medicine, Nippon Veterinary and Life Science University, Musashino-shi, Tokyo 180-8602, Japan.

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Daisuke Hasegawa Department of Clinical Veterinary Medicine, Nippon Veterinary and Life Science University, Musashino-shi, Tokyo 180-8602, Japan.

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Yuji Hamamoto Department of Clinical Veterinary Medicine, Nippon Veterinary and Life Science University, Musashino-shi, Tokyo 180-8602, Japan.

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Shunta Mizoguchi Department of Clinical Veterinary Medicine, Nippon Veterinary and Life Science University, Musashino-shi, Tokyo 180-8602, Japan.

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Takayuki Kuwabara Department of Clinical Veterinary Medicine, Nippon Veterinary and Life Science University, Musashino-shi, Tokyo 180-8602, Japan.

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Aki Fujiwara-Igarashi Department of Clinical Veterinary Medicine, Nippon Veterinary and Life Science University, Musashino-shi, Tokyo 180-8602, Japan.

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Masaya Tsuboi Laboratory of Veterinary Pathology, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Bunkyo-ku, Tokyo 113-8657, Japan.

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James Ken Chambers Laboratory of Veterinary Pathology, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Bunkyo-ku, Tokyo 113-8657, Japan.

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Michio Fujita Department of Clinical Veterinary Medicine, Nippon Veterinary and Life Science University, Musashino-shi, Tokyo 180-8602, Japan.

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Kazuyuki Uchida Laboratory of Veterinary Pathology, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Bunkyo-ku, Tokyo 113-8657, Japan.

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Abstract

OBJECTIVE To investigate epilepsy-related neuropathologic changes in cats of a familial spontaneous epileptic strain (ie, familial spontaneous epileptic cats [FSECs]).

ANIMALS 6 FSECs, 9 age-matched unrelated healthy control cats, and 2 nonaffected (without clinical seizures)dams and 1 nonaffected sire of FSECs.

PROCEDURES Immunohistochemical analyses were used to evaluate hippocampal sclerosis, amygdaloid sclerosis, mossy fiber sprouting, and granule cell pathological changes. Values were compared between FSECs and control cats.

RESULTS Significantly fewer neurons without gliosis were detected in the third subregion of the cornu ammonis (CA) of the dorsal and ventral aspects of the hippocampus as well as the central nucleus of the amygdala in FSECs versus control cats. Gliosis without neuronal loss was also observed in the CA4 subregion of the ventral aspect of the hippocampus. No changes in mossy fiber sprouting and granule cell pathological changes were detected. Moreover, similar changes were observed in the dams and sire without clinical seizures, although to a lesser extent.

CONCLUSIONS AND CLINICAL RELEVANCE Findings suggested that the lower numbers of neurons in the CA3 subregion of the hippocampus and the central nucleus of the amygdala were endophenotypes of familial spontaneous epilepsy in cats. In contrast to results of other veterinary medicine reports, severe epilepsy-related neuropathologic changes (eg, hippocampal sclerosis, amygdaloid sclerosis, mossy fiber sprouting, and granule cell pathological changes) were not detected in FSECs. Despite the use of a small number of cats with infrequent seizures, these findings contributed new insights on the pathophysiologic mechanisms of genetic-related epilepsy in cats.

Abstract

OBJECTIVE To investigate epilepsy-related neuropathologic changes in cats of a familial spontaneous epileptic strain (ie, familial spontaneous epileptic cats [FSECs]).

ANIMALS 6 FSECs, 9 age-matched unrelated healthy control cats, and 2 nonaffected (without clinical seizures)dams and 1 nonaffected sire of FSECs.

PROCEDURES Immunohistochemical analyses were used to evaluate hippocampal sclerosis, amygdaloid sclerosis, mossy fiber sprouting, and granule cell pathological changes. Values were compared between FSECs and control cats.

RESULTS Significantly fewer neurons without gliosis were detected in the third subregion of the cornu ammonis (CA) of the dorsal and ventral aspects of the hippocampus as well as the central nucleus of the amygdala in FSECs versus control cats. Gliosis without neuronal loss was also observed in the CA4 subregion of the ventral aspect of the hippocampus. No changes in mossy fiber sprouting and granule cell pathological changes were detected. Moreover, similar changes were observed in the dams and sire without clinical seizures, although to a lesser extent.

CONCLUSIONS AND CLINICAL RELEVANCE Findings suggested that the lower numbers of neurons in the CA3 subregion of the hippocampus and the central nucleus of the amygdala were endophenotypes of familial spontaneous epilepsy in cats. In contrast to results of other veterinary medicine reports, severe epilepsy-related neuropathologic changes (eg, hippocampal sclerosis, amygdaloid sclerosis, mossy fiber sprouting, and granule cell pathological changes) were not detected in FSECs. Despite the use of a small number of cats with infrequent seizures, these findings contributed new insights on the pathophysiologic mechanisms of genetic-related epilepsy in cats.

Epilepsy is a common chronic and functional cerebral disorder in both veterinary and human medicine. The hippocampus is the most widely studied brain region for epilepsy.1 In human medicine, hippocampal sclerosis is the most frequent histopathologic finding encountered in patients undergoing surgical treatment for MTLE.2,3 Hippocampal sclerosis is defined as selective neuronal cell loss with concomitant astrogliosis in the hippocampal formation.3 In addition, granule cell pathological changes and mossy fiber sprouting are often observed in the hippocampus in association with hippocampal sclerosis.3–7

The amygdala is thought to play a role in the generation and propagation of epileptic seizures in TLE. Neuropathologic studies8,9 have revealed neuronal loss and gliosis in the amygdala (referred to as amygdaloid sclerosis) of human patients with TLE. Pathological changes of the epileptic brain in veterinary medicine have been described anecdotally in case reports; however, hippocampal sclerosis and granule cell pathological changes have been reported10 in a large cohort of cats with epilepsy.

Cats of a familial spontaneous epileptic strain (ie, FSECs) are the only epileptic cats with a suspected genetic cause and apparent FMTLE11; a colony of these cats has been established and maintained at Nippon Veterinary and Life Science University. The FSEC strain consists of cats with recurrent seizures and several nonaffected (ie, without clinical seizures) dams and sires of the first generation of FSECs. Clinical signs in FSECs are of 2 seizure types: spontaneous limbic seizures with or without secondary generalizations and vestibular stimulation–induced generalized tonic-clonic seizures. Seizures in FSECs are similar to those in cats with amygdaloid or hippocampal (or both) kindling or kainic acid12–14 and similar to vestibular stimulation–induced generalized seizures in EL mice (a strain of mice with genetic epilepsy).15 Furthermore, spontaneous limbic seizures with or without secondary generalizations are the common form of epilepsy in cats.16,a Indeed, FSECs have interictal spikes, which are predominantly distributed in the unilateral or bilateral temporal regions (or both) and are generally synchronized in scalp EEGs.11,17 In addition, intracranial EEG with video monitoring has revealed seizure activities in the amygdala or hippocampus (or both) with or without onset of clinical seizures.17 In that study,17 intracranial EEG was performed on 5 FSECs, and subsequent histologic evaluation of tissues from those cats revealed lower cell numbers in the third subregion of the CA (CA3). However, implanted depth electrodes were used for intracranial EEG in the hippocampus and neuronal cell counting was performed only for the CA1 through CA3 subregions. Because of those limitations, many neuropathologic features of FSECs may have remained uncharacterized. Although typical hippocampal sclerosis findings were not evident by use of conventional MRI in FSECs of another study,18 hippocampal volumetry determined with MRI revealed atrophic or asymmetric hippocampi in those FSECs. Additionally, results of a study19 involving MRI determination of interictal diffusion and perfusion suggested there were microscopic structural changes in the hippocampus and amygdala. Therefore, we hypothesized that microstructural abnormalities in the hippocampus or amygdala (or both) could represent structurally abnormal zones in FSECs.

The objective of the study reported here was to noninvasively evaluate microstructural alterations in the hippocampus and amygdala of FSECs. Therefore, we investigated epilepsy-related pathological changes such as hippocampal sclerosis, amygdaloid sclerosis, mossy fiber sprouting, and granule cell pathological changes in FSECs and compared them with results for clinically normal control cats.

Materials and Methods

Animals

Three groups of cats were included in the study. The study, including the care of the cats and maintenance of the FSEC colony, was approved by the Animal Care and Use Committee and the Bioethics Committee of Nippon Veterinary and Life Science University (accession Nos. 26K-29, 27K-10, and 28K-4).

Six FSECs (3 that had recurrent spontaneous limbic seizures and 3 that had vestibular stimulation–induced generalized seizures) were used for the study; these cats had been included previously in other studies.17–19 The cats had sporadic or rare (1 or 2 seizures/y) seizures, as determined by use of a 24-hour video monitoring system for > 1 year. Generally, seizures terminated within 1 to 2 minutes and no medical treatment was required because of their lack of severity and relative infrequency. Median age at which these cats had the initial seizure was 8.5 months (range, 3 to 16 months), and median duration of epilepsy was 41.4 months (range, 7 to 87 months). It was possible that the FSECs also had subclinical seizures. All 6 FSECs had interictal spikes evident on scalp EEG, which predominantly arose from the temporal region. The EEG abnormalities were bilateral in 3 FSECs and unilateral in the other 3 FSECs. Hippocampal asymmetry was observed by use of MRI volumetry in 4 FSECs (2 had left-sided hippocampal atrophy and 2 had right-sided hippocampal atrophy). The FSECs comprised 3 males and 3 females. Median age of FSECs was 52.5 months (range, 15 to 96 months), and median body weight was 3.8 kg (range, 3.4 to 4.4 kg).

Nine age-matched healthy cats (3 males and 6 females) without any history of seizures or neurologic abnormalities and that were not related to the FSECs comprised a control group. Median age of the control cats was 81.0 months (range, 31 to 138 months), which did not differ significantly (P = 0.175; Mann-Whitney U test) from that of the FSECs. Median body weight was 3.4 kg (range, 3.2 to 3.8 kg).

Finally, 3 nonaffected cats (ie, without clinical seizures; 2 dams and 1 sire) of the first generation of FSECs were also included. Median age was 130 months (range, 108 to 142 months), and median body weight was 3.3 kg (range, 2.5 to 4.2 kg).

Histologic examination and immunohistochemical analysis

All cats were euthanized by IV administration of an overdose of pentobarbital (100 mg/kg). Subsequently, the entire brain of all 6 FSECs, 3 healthy control cats, and the 2 nonaffected dams and 1 nonaffected sire were immediately fixed in neutral-buffered 10% formalin. For the remaining 6 control cats, 1 hemisphere of the brain was immediately fixed in neutral-buffered 10% formalin for use in the study reported here, and the other hemisphere was cryopreserved for use in future studies.

After fixation was complete, brains were sectioned transversely at the level of the frontal lobe, striatum, amygdala, hippocampus, midbrain, medulla oblongata, and cerebellum. All brain segments were dehydrated in an ascending series of ethanol solutions, which was followed by a xylene solution. Tissues were embedded in paraffin and cut into 4-μm-thick slices.

Staining with H&E and Kluver-Barrera stains was performed by use of conventional methods. Immunohistochemical analysis was performed with a neuron-specific nuclear protein (ie, NeuN), GFAP, and dynorphin A to investigate neuron numbers and gliosis in the hippocampus and amygdala, granule cell pathological changes, and mossy fiber sprouting. Tissue sections were deparaffinized; sections were placed in 10mM citrate buffer solution and autoclaved at 120°C for 10 minutes for antigen retrieval, except for sections used for GFAP staining. Subsequently, sections were incubated with 3% hydrogen peroxide in methanol for 30 minutes to deactivate endogenous peroxidases, which was followed by incubation with 5% skim milk in Tris-buffered saline solution at 37°C for 30 minutes to decrease nonspecific antibody binding. Sections then were incubated with mouse anti-NeuNb (dilution, 1:500), rabbit anti-GFAPc (dilution, 1:400), or rabbit anti–dynorphin A antibodyd (dilution, 1:500) at 37°C for 40 minutes. A horseradish peroxidase–labeled polymere was used as the secondary antibody. Reactions were developed by use of 3,3′-diaminobenzidine tetrachloridef with hydrogen peroxide in a tris–hydrochloric acid buffer. Sections stained with anti-NeuN were counterstained with methyl green stain, whereas the remaining sections were counterstained with Mayer hematoxylin stain.

Subregions CA1 through CA4 of the hippocampus were evaluated.20 Number of neurons, gliosis, granule cell pathological changes, and mossy fiber sprouting in the hippocampus were evaluated. The amygdala is a heterogeneous collection of nuclei, and the feline amygdala consists of the basolateral, basomedial, lateral, central, cortical, and medial nuclei. We used subnuclei mapping and nomenclature described in another study.21 However, it can be difficult to accurately ascertain each area of the amygdala.22 Therefore, we focused on assessing the number of neurons and gliosis at the medial divisions of 3 amygdaloid nuclei that were easy to visually examine (ie, central nucleus, lateral nucleus, and basolateral nucleus), as described elsewhere.21 In addition, all specimens, including slides containing regions other than the hippocampus and amygdala, were reviewed for the presence of focal cortical dysplasia and other abnormalities (eg, tectonic hippocampal malformations23 or ectopic neuronal clusters24,25). Digital images of each stained specimen were obtained by use of a microscopeg equipped with a digital camerah and associated software.i An image-processing programj was used for image analysis.

Neuronal nuclei with positive results for staining with NeuN were counted with a 100X objective by applying manual tags in subfields (830 × 664 μm) projected on a computer monitor by use of the image-processing program.j Source of samples was concealed from investigators during quantification. The NeuN-immunoreactive cell bodies were tagged on the computer screen and counted separately within each of the hippocampal subregions (CA1 through CA4), the granular cell layer of the dentate gyrus, and the 3 amygdaloid nuclei (central nucleus, lateral nucleus, and basolateral nucleus). Cells were counted 4 times in each region. In addition, quantification of CA4 involved use of 2 nonoverlapping regions of interest (each was 300 × 300 μm and was within the 830 × 664-μm subfield) selected in each processed image. This allowed us to specifically quantify CA4 because some images included structures other than the CA4. Except for CA4, all cell counts were obtained by calculating the mean of the total number of NeuN-immunoreactive neurons for each specimen; the number of neurons per unit area (0.55 mm2) then was calculated. The unit area for CA4 corresponded to the 2 regions of interest (0.18 mm2), and the mean of the total cell number was determined per unit.

Gliosis was quantitatively evaluated by determining the area of GFAP immunoreactivity by use of the red-green-blue measure of the imaging software.26,27 Gliosis was evaluated in CA1 through CA3 in GFAP-stained specimens (830 × 664 μm) by use of a 100X objective. Immunoreactivity was evaluated, and 2 nonoverlapping regions of interest (each was 300 × 300 μm and was within the 830 × 664-μm subfield) were chosen in CA4 to avoid the CA3 and dentate gyrus in each image.

Mossy fiber sprouting was evaluated on dynorphin A–stained specimens by use of a grading system adapted from a method used for human samples.4,28 Grade 0 corresponded to a normal pattern of the mossy fiber pathway in CA3 and CA4, grade 1 corresponded to focal mossy fiber sprouting in the inner molecular layer, and grade 2 corresponded to a wide band of mossy fiber sprouting throughout the molecular layer.

For human medicine, there is not an international consensus concerning granule cell pathological changes, although neuropathologic criteria have been suggested, which include an increase in granule cell lamination (> 10 layers),29 dispersion, ectopic cells, clusters, and bilamination.5 In the study reported here, classification and definition of granule cell pathological changes described elsewhere5 were used for the evaluation of the dentate gyrus. The dentate gyrus was subdivided into 3 regions, each of which represented one-third of the structure: the mesial part of the dentate gyrus (also called the internal limb), the lateral part of the dentate gyrus (also called the external limb) located between CA2 and CA4, and the middle area located between the internal and external limbs.5,30 Thickness of the granular cell layer, which was reflected by the number of cell layers on a straight line, was measured in 3 noncurved areas in the internal and external limbs of the NeuN-stained specimens. Mean measured thickness and mean number of granular cell layers for each of these 3 areas were used for statistical analysis.

Statistical analysis

The dorsal and ventral aspects of the hippocampus were evaluated separately. We did not make statistical comparisons between the 2 dams and 1 sire and the other 2 groups. Instead, findings for the 2 dams and 1 sire were used as supporting evidence when considering results of comparisons between the FSECs and control cats.

Therefore, only the FSECs and control cats were statistically compared. Data were expressed as median and interquartile range. All statistical analyses between FSECs and control cats were conducted by use of Mann-Whitney U tests with statistical software.k Findings were considered significant at values of P < 0.05.

Results

Histologic examination and immunohistochemical analysis

Investigation of the entire brain, except for sections of the hippocampus and amygdala stained with the H&E and Kluver-Barrera stains, revealed no obvious structural brain abnormalities (eg, focal cortical dysplasia) for any groups. In the hippocampus, there were no abnormal neuronal clusters, disorganized neuronal cells, or structural anomalies of the pyramidal cell layer, which have been described in Ihara epileptic rats (rats with genetic limbic-like seizures),31 rats exposed to methylazoxymethanol acetates,24 and certain human patients with TLE.32

Significantly fewer NeuN-positive neurons were observed in the CA3 of the dorsal (P = 0.010) and ventral (P = 0.002) aspects of the hippocampus of FSECs, compared with results for the control cats (Figure 1). In contrast to results for the CA3, there were no significant differences between FSECs and control cats for CA1, CA2, and CA4 of the dorsal and ventral aspects of the hippocampus. There was no clear pathological difference in the hippocampus among individual FSECs. Additionally, there was an apparent pattern of fewer neurons when comparing the number of neurons of the CA3 for the dorsal and ventral aspects of the hippocampus of FSECs, control cats, and the 2 dams and 1 sire of the first generation of FSEC cats with the number of neurons in the central nucleus of the amygdala (Figure 2).

Figure 1—
Figure 1—

Photomicrographs of tissues after immunohistochemical staining with NeuN for the entire hippocampus (A and B) and the ventral aspect of the CA3 subregion of the hippocampus (C and D) from a representative control cat (A and C) and a representative FSEC (B and D) and box-and-whisker plots of cells with positive staining for NeuN in subregion CA1 (E and I), CA2 (F and J), CA3 (G and K), and CA4 (H and L) of the dorsal (E through H) and ventral (I through L) aspects of the hippocampus of 9 control cats (white boxes) and 6 FSECs (gray boxes). In the photomicrographs, there were no obvious morphological abnormalities in the pyramidal cell layer (arrowheads) or dentate gyrus (arrows), and neuronal density was less in the FSEC than in the control cat. No dispersion of granule cells was evident. Bar = 1,000 μm for panels A and B and 100 μm for panels C and D. In the box-and-whisker plots, each box represents the quartile range, the horizontal bar is the median, the whiskers are the maximum and minimum values excluding outliers, and the circles are outliers. Notice that the scale on the y-axis differs among panels. *†Within a subregion within the dorsal or ventral aspect of the hippocampus, value differs significantly (*P < 0.05; P = 0.01) from the value for the control cats.

Citation: American Journal of Veterinary Research 79, 3; 10.2460/ajvr.79.3.324

Figure 2—
Figure 2—

Box-and-whisker plots of the number of neurons in the CA3 subregion in the dorsal (A) and ventral (B) aspects of the hippocampus and the central nucleus of the amygdala (C) of 9 control cats (white boxes), 6 FSECs (gray boxes), and 2 nonaffected (ie, without clinical seizures) dams and 1 nonaffected sire of the first generation of FSECs (diagonal-striped boxes). Notice that the scale on the y-axis differs among the panels. See Figure 1 for remainder of key.

Citation: American Journal of Veterinary Research 79, 3; 10.2460/ajvr.79.3.324

To evaluate the association of the number of neurons with side of the EEG or MRI volumetric atrophy, the number of neurons for the CA3 of each side was compared with the results for each side in each cat. However, there was no obvious association between side of the EEG or MRI hippocampal volumetric atrophy with the side with a lower number of neurons in the CA3 (data not shown).

Comparison of FSECs and control cats revealed a significant (P = 0.015) difference in hippocampal GFAP immunoreactivity. This was observed in the CA4 of the ventral aspect of the hippocampus but not in the other hippocampal subregions (Figure 3).

Figure 3—
Figure 3—

Photomicrographs of tissues after immunohistochemical staining with GFAP for the entire hippocampus (A and B) and the ventral aspect of the CA3 subregion of the hippocampus (C and D) from a representative control cat (A and C) and a representative FSEC (B and D) and box-and-whisker plots of the immunoreactivity for GFAP in subregion CA1 (E and I), CA2 (F and J), CA3 (G and K), and CA4 (H and L) of the dorsal (E through H) and ventral (I through L) aspects of the hippocampus of 9 control cats (white boxes) and 6 FSECs (gray boxes; E through L). Bar = 1,000 μm for panels A and B and 100 μm for panels C and D. Notice that the scale on the y-axis differs among panels. See Figure 1 for remainder of key.

Citation: American Journal of Veterinary Research 79, 3; 10.2460/ajvr.79.3.324

Furthermore, significantly (P < 0.001) fewer neurons in the central nucleus of the amygdala were found in FSECs than in control cats, but there were no significant differences in the number of neurons in the basolateral nucleus and lateral nucleus of the amygdala between the 2 groups (Figure 4). There was no clear pathological difference in the amygdala among individual FSECs. In addition, no significant differences in GFAP immunoreactivity were observed in any nucleus of the amygdala. Moreover, there also seemed to be an apparent pattern for fewer neurons within the central nucleus of the amygdala of FSECs than in the 2 dams and 1 sire (Figure 2).

Figure 4—
Figure 4—

Photomicrographs of tissues after immunohistochemical staining with NeuN for the entire amygdala (A and B) and the central nucleus (CE), lateral nucleus (LA), and basolateral nucleus (BLA) of the amygdala (C and D) from a representative control cat (A and C) and a representative FSEC (B and D) and box-and-whisker plots of the number of neurons (E through G) with positive staining for NeuN and the immunoreactivity for GFAP (H through J) in the basolateral nucleus (E and H), lateral nucleus (F and I), and central nucleus (G and J) of the amygdala of 9 control cats (white boxes) and 6 FSECs (gray boxes). Bar = 1,000 μm for panels A and B and 100 μm for panels C and D. Notice that the scale on the y-axis differs among panels. See Figure 1 for remainder of key.

Citation: American Journal of Veterinary Research 79, 3; 10.2460/ajvr.79.3.324

There was strong staining for dynorphin A in the CA4 and CA3, and the staining was gradually reduced toward CA1 (Figure 5). All cats had a grade of 0 for mossy fiber sprouting. None of the FSECs and control cats had dynorphin A staining in the molecular layer, except at the tip of the septal and temporal poles of the hippocampus.

Figure 5—
Figure 5—

Photomicrographs of tissues after immunohistochemical staining with dynorphin A for the entire hippocampus (A and B) and the ventral aspect of the hippocampus (C and D) from a representative control cat (A and C) and a representative FSEC (B and D). No mossy fiber sprouting was observed. Bar = 1,000 μm for panels A and B and 100 μm for panels C and D.

Citation: American Journal of Veterinary Research 79, 3; 10.2460/ajvr.79.3.324

No significant differences in granule cell pathological change were detected between FSECs and control cats with regard to median measured thickness and number of granule cell layers. Similarly, no changes in variables such as ectopic cells, bilamination, thinning, gaps, clusters, or granule cell dispersion were observed in any of the cats (Figure 1). Median values for measured thickness and number of granule cell layers were determined (Table 1).

Table 1—

Median (quartile deviation) values for the measured thickness (μm) and number of granule cell layers in the dorsal and ventral regions of the lateral part (also called the external limb) and mesial part (also called the internal limb) of the dentate gyrus in 6 FSECs and 9 control cats.

  External limbInternal limb
GroupRegionThicknessNo. of layersThicknessNo. of layers
FSECDorsal76.81 (6.82)6.33 (0.41)77.73 (5.79)6.67 (0.54)
 Ventral71.48 (3.96)5.50 (0.50)70.30 (3.18)5.67 (0.50)
ControlDorsal73.65 (2.62)6.33 (0.08)65.36 (5.35)6.17 (0.38)
 Ventral70.28 (7.00)6.00 (0.38)69.67 (5.86)5.67 (0.33)

Values did not differ significantly (P ≥ 0.05) between groups.

Discussion

Pathological changes of the hippocampus in cats with epilepsy have been reported.10,33–39 Authors of most of these reports33–37,39 described cats with hippocampal necrosis, whereas authors of 1 report10 referred to pathological changes in cats with idiopathic epilepsy. In contrast to reports of hippocampal sclerosis or hippocampal necrosis in cats, we found significantly fewer neurons without gliosis in the CA3 of the dorsal and ventral aspects of the hippocampus of FSECs. Although fewer numbers of neurons without gliosis in the CA3 might be caused by a genetic drift in the FSEC colony, this subtle hippocampal change could be one of the factors that causes epileptic seizures. Authors of 1 study2 of humans with FMTLE reported patterns of cell loss similar to those for sporadic MTLE, whereas other authors reported no evidence of neuronal loss in the pyramidal cell layer or dentate gyrus in a patient with FMTLE.40 Considering results of these previous reports and results of the present study, it is clear that FMTLE is a heterogeneous disorder from the point of view of clinical and microscopic findings. Furthermore, significantly fewer neurons without gliosis were detected in the central nucleus of the amygdala of FSECs. To our knowledge, there have been no reports of amygdaloid pathological changes in cats with naturally occurring epilepsy. It has been reported that the findings were those seen (eg, neuronal loss in the lateral nucleus of the amygdala) in human patients with TLE9,22,41 and sudden unexpected death of humans attributable to epilepsy42 in addition to neuronal loss in the basolateral nucleus43 and the complex44 of the amygdala in animals with experimentally induced status epilepticus, and they are thought to be the result of severe epileptic seizures. In contrast, FSECs of the study reported here had fewer neurons in the central nucleus of the amygdala and CA3 of the hippocampus, compared with those of healthy control cats.

The FSECs were considered likely to have fewer neurons in the CA3 of the hippocampus and central nucleus of the amygdala, which could partially have been associated with seizure generation. Investigators have concluded that in humans, families with epilepsy, including healthy family members, express a subtle, genetically determined, preexisting developmental malformation in the hippocampus.32,45 It has been suggested46,47 that inherited hippocampal abnormalities in humans would not necessarily lead to epilepsy. In the present study, the nonaffected (ie, without clinical seizures) dams and sire of FSECs also had an apparent pattern of fewer neurons in the CA3 of the hippocampus and central nucleus of the amygdala. Therefore, results for the study reported here suggested that the lower number of neurons in the hippocampus and amygdala may have been an endophenotype of FSECs, which may have been associated with seizure susceptibility. This FSEC endophenotype could support the notion that FMTLE involves a major gene that leads to hippocampal and amygdaloid anomalies and that this phenotype could be influenced by additional genetic or environmental factors.48

In addition to the aforementioned findings of fewer neurons, gliosis was limited to the CA4 in FSECs, which corresponded to the category of “no hippocampal sclerosis–gliosis only” described in the international consensus for human hippocampal sclerosis.3 In human medicine, this type of hippocampal pathological change is reportedly seen in up to 20% of TLE cases. The finding equivalent to the no hippocampal sclerosis–gliosis only category was reportedly detected in EL mice, a condition that serves as a natural model of TLE.49 However, it may not be simple to conclude that no hippocampal sclerosis–gliosis only is a cause or effect in the same manner that hippocampal sclerosis in humans has been established as both the cause and an effect of epilepsy.50 In contrast to the aforementioned fewer neurons that were observed in both FSECs and the 2 dams and 1 sire, the fact that a significant difference was observed only for FSECs would suggest that this lesion is related to epilepsy. Gliosis in the CA4 was only observed in the ventral aspect of the hippocampus, which is consistent with the fact that the ventral aspect of the hippocampus is the homolog of the hippocampal head in humans,51 which is the area most commonly affected by epilepsy-associated changes in humans.52 Therefore, although it was impossible to confirm that the no hippocampal sclerosis–gliosis only finding was the cause or a result of epileptic seizures, we speculate that gliosis in the CA4 of FSECs may be the result of recurrent seizures.

Hippocampal sclerosis or its associated changes (ie, granule cell pathological changes and mossy fiber sprouting) were not detected in cats of the study reported here. In the dentate gyrus in the ventral aspect of the hippocampus, the length of both external and internal limbs was approximately 70 μm, which is consistent with a previously reported length of granule cell layers in cats.l An association between granule cell pathological changes and the degree of hippocampal sclerosis has been reported in humans6 and cats.10 It was also reported that the grade of dynorphin A immunoreactivity is higher (including more severe hippocampal sclerosis) with granule cell pathological changes.7 Those reports could explain the reason that the FSECs of the present study did not have granule cell pathological changes or mossy fiber sprouting.

Although substantial bilateral hippocampal atrophy was not observed, a previous study18 conducted by our research group revealed significant asymmetry in the hippocampal volume of FSECs, which indicated the likelihood of hippocampal atrophy in those cats. Loss of hippocampal volume is associated with cell loss but not with seizure frequency.53 Although 4 of 6 FSECs in the present study had hippocampal asymmetry as determined by use of MRI volumetry, we did not detect an obvious association of these asymmetries in 1 side of the CA3 with the lower number of neurons or the EEG findings. Interestingly, it recently has been reported54 that extracellular matrix molecules are important for maintenance of the hippocampal volume. Although FSECs did not have substantially fewer neuronal cells in areas other than the CA3, factors other than the number of neurons (eg, extracellular matrix) may also be associated with hippocampal volume. Additionally, the total number of hippocampal neurons was not determined in the present study, whereas the MRI volumetry measured the entire hippocampal volume. Therefore, we cannot draw clear conclusions about the relevance of these findings.

The present study had some limitations. First, only FSECs that had infrequent seizures were included. Considering the value of the FSEC colony as the only cats with suspected genetic epilepsy in the world, it was impossible to preferentially euthanize cats with a higher frequency of seizures to enable us to maintain the colony. Furthermore, the number of FSECs available for the present study was limited. The study reported here required that histologic examinations of several factors (eg, hippocampal sclerosis, granule cell pathological changes, and mossy fiber sprouting) be conducted. Therefore, the isotropic fractionator method for neuronal counting was not used because that method results in destruction of the tissue.55 However, the neuronal marker (NeuN) used in the present study is a well-established marker,56 and manual counting could be easily performed via visual examination. Although we cannot exclude the possibility that slight counting errors might have occurred because we did not use stereological procedures, a convenient and easy manual counting method that involved NeuN immunohistochemical analysis was used.

The pathophysiologic processes of idiopathic or genetic epilepsy of cats remain unclear. Although FSECs did not have severe epilepsy-related changes (eg, hippocampal sclerosis, granule cell pathological changes, and mossy fiber sprouting), the study reported here highlighted the pathological character of a familial form of epilepsy in cats. It is important to know that hippocampal and amygdaloid abnormalities exist in FSECs, even those with infrequent seizures. To our knowledge, there have been no previous studies conducted to address the pathological findings for cats with genetic epilepsy or suspected genetic epilepsy. It is important to evaluate epilepsy with a mild clinical course in veterinary medicine to enable an understanding of the pathophysiologic mechanisms of epilepsy. Therefore, results for the present study may provide additional insights about epileptic animals.

Acknowledgments

Supported in part by a grant from the Japan Epilepsy Research Foundation (2015).

Presented in abstract form at the 50th Congress of The Japan Epilepsy Society, Shizuoka, Japan, October 2016.

The authors thank Dr. Dai Nagakubo for assistance with collection of brain tissue samples from 6 control cats.

ABBREVIATIONS

CA

Cornu ammonis

EEG

Electroencephalography

FMTLE

Familial mesial temporal lobe epilepsy

FSEC

Familial spontaneous epileptic cat

GFAP

Glial fibrillary acidic protein

MTLE

Mesial temporal lobe epilepsy

NeuN

Neuron-specific nuclear protein

TLE

Temporal lobe epilepsy

Footnotes

a.

Cizinauskas S, Fatzer R, Schenkel M, et al. Can idiopathic epilepsy be confirmed in cats? (abstr). J Vet Intern Med 2003;17:246.

b.

Chemicon International, Temecula, Calif.

c.

DAKO, Carpinteria, Calif.

d.

Phoenix Pharmaceuticals Inc, Burlingame, Calif.

e.

EnVision+ System, DAKO, Carpinteria, Calif.

f.

Dojindo Laboratories, Kumamoto, Japan.

g.

BX 50, Olympus, Tokyo, Japan.

h.

DXM 1200F, Nikon, Tokyo, Japan.

i.

ACT-1 software, Nikon, Tokyo, Japan.

j.

ImageJ, National Institutes of Health, Bethesda, Md. Available at: imagej.nih.gov/ij/. Accessed Apr 3, 2015.

k.

EZR, Saitama Medical Center, Saitama, Japan.

l.

Wagner E, Rosati M, Fischer A, et al. Granule cell dispersion in cats with and without epilepsy (abstr). J Vet Intern Med 2014;29:1451.

References

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    • Crossref
    • Search Google Scholar
    • Export Citation
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    • Crossref
    • Search Google Scholar
    • Export Citation
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    • Crossref
    • Search Google Scholar
    • Export Citation
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    • Crossref
    • Search Google Scholar
    • Export Citation
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    • Search Google Scholar
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    • Search Google Scholar
    • Export Citation
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    • Search Google Scholar
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    • Export Citation
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    • Search Google Scholar
    • Export Citation
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    • Export Citation
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    • Search Google Scholar
    • Export Citation
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    • Crossref
    • Search Google Scholar
    • Export Citation
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    • Crossref
    • Search Google Scholar
    • Export Citation
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    • Crossref
    • Search Google Scholar
    • Export Citation
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    • Search Google Scholar
    • Export Citation
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    • Search Google Scholar
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    • Crossref
    • Search Google Scholar
    • Export Citation
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    • Crossref
    • Search Google Scholar
    • Export Citation
  • 30. Pauli E, Hildebrandt M, Romstöck J, et al. Deficient memory acquisition in temporal lobe epilepsy is predicted by hippocampal granule cell loss. Neurology 2006;67:13831389.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 31. Tsuji A, Amano S, Yokoyama M, et al. Neuronal microdysgenesis and acquired lesions of the hippocampal formation connected with seizure activities in Ihara epileptic rat. Brain Res 2001;901:111.

    • Crossref
    • Search Google Scholar
    • Export Citation
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  • 33. Fatzer R, Gandini G, Jaggy A, et al. Necrosis of hippocampus and piriform lobe in 38 domestic cats with seizures: a retrospective study on clinical and pathologic findings. J Vet Intern Med 2000;14:100104.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 34. Brini E, Gandini G, Crescio I, et al. Necrosis of hippocampus and piriform lobe: clinical and neuropathological findings in two Italian cats. J Feline Med Surg 2004;6:377381.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 35. Schmied O, Scharf G, Hilbe M, et al. Magnetic resonance imaging of feline hippocampal necrosis. Vet Radiol Ultrasound 2008;49:343349.

    • Crossref
    • Search Google Scholar
    • Export Citation
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    • Crossref
    • Search Google Scholar
    • Export Citation
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    • Crossref
    • Search Google Scholar
    • Export Citation
  • 38. Klang A, Thaller D, Schmidt P, et al. Bilateral dentate gyrus structural alterations in a cat associated with hippocampal sclerosis and intraventricular meningioma. Vet Pathol 2015;52:11831186.

    • Crossref
    • Search Google Scholar
    • Export Citation
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  • Figure 1—

    Photomicrographs of tissues after immunohistochemical staining with NeuN for the entire hippocampus (A and B) and the ventral aspect of the CA3 subregion of the hippocampus (C and D) from a representative control cat (A and C) and a representative FSEC (B and D) and box-and-whisker plots of cells with positive staining for NeuN in subregion CA1 (E and I), CA2 (F and J), CA3 (G and K), and CA4 (H and L) of the dorsal (E through H) and ventral (I through L) aspects of the hippocampus of 9 control cats (white boxes) and 6 FSECs (gray boxes). In the photomicrographs, there were no obvious morphological abnormalities in the pyramidal cell layer (arrowheads) or dentate gyrus (arrows), and neuronal density was less in the FSEC than in the control cat. No dispersion of granule cells was evident. Bar = 1,000 μm for panels A and B and 100 μm for panels C and D. In the box-and-whisker plots, each box represents the quartile range, the horizontal bar is the median, the whiskers are the maximum and minimum values excluding outliers, and the circles are outliers. Notice that the scale on the y-axis differs among panels. *†Within a subregion within the dorsal or ventral aspect of the hippocampus, value differs significantly (*P < 0.05; P = 0.01) from the value for the control cats.

  • Figure 2—

    Box-and-whisker plots of the number of neurons in the CA3 subregion in the dorsal (A) and ventral (B) aspects of the hippocampus and the central nucleus of the amygdala (C) of 9 control cats (white boxes), 6 FSECs (gray boxes), and 2 nonaffected (ie, without clinical seizures) dams and 1 nonaffected sire of the first generation of FSECs (diagonal-striped boxes). Notice that the scale on the y-axis differs among the panels. See Figure 1 for remainder of key.

  • Figure 3—

    Photomicrographs of tissues after immunohistochemical staining with GFAP for the entire hippocampus (A and B) and the ventral aspect of the CA3 subregion of the hippocampus (C and D) from a representative control cat (A and C) and a representative FSEC (B and D) and box-and-whisker plots of the immunoreactivity for GFAP in subregion CA1 (E and I), CA2 (F and J), CA3 (G and K), and CA4 (H and L) of the dorsal (E through H) and ventral (I through L) aspects of the hippocampus of 9 control cats (white boxes) and 6 FSECs (gray boxes; E through L). Bar = 1,000 μm for panels A and B and 100 μm for panels C and D. Notice that the scale on the y-axis differs among panels. See Figure 1 for remainder of key.

  • Figure 4—

    Photomicrographs of tissues after immunohistochemical staining with NeuN for the entire amygdala (A and B) and the central nucleus (CE), lateral nucleus (LA), and basolateral nucleus (BLA) of the amygdala (C and D) from a representative control cat (A and C) and a representative FSEC (B and D) and box-and-whisker plots of the number of neurons (E through G) with positive staining for NeuN and the immunoreactivity for GFAP (H through J) in the basolateral nucleus (E and H), lateral nucleus (F and I), and central nucleus (G and J) of the amygdala of 9 control cats (white boxes) and 6 FSECs (gray boxes). Bar = 1,000 μm for panels A and B and 100 μm for panels C and D. Notice that the scale on the y-axis differs among panels. See Figure 1 for remainder of key.

  • Figure 5—

    Photomicrographs of tissues after immunohistochemical staining with dynorphin A for the entire hippocampus (A and B) and the ventral aspect of the hippocampus (C and D) from a representative control cat (A and C) and a representative FSEC (B and D). No mossy fiber sprouting was observed. Bar = 1,000 μm for panels A and B and 100 μm for panels C and D.

  • 1. Thom M. Review: hippocampal sclerosis in epilepsy: a neuropathology review. Neuropathol Appl Neurobiol 2014;40:520543.

  • 2. Andrade-Valença LP, Valença MM, Velasco TR, et al. Mesial temporal lobe epilepsy: clinical and neuropathologic findings of familial and sporadic forms. Epilepsia 2008;49:10461054.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 3. Blümcke I, Thom M, Aronica E, et al. International consensus classification of hippocampal sclerosis in temporal lobe epilepsy: a Task Force report from the ILAE Commission on Diagnostic Methods. Epilepsia 2013;54:13151329.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 4. Bandopadhyay R, Liu JY, Sisodiya SM, et al. A comparative study of the dentate gyrus in hippocampal sclerosis in epilepsy and dementia. Neuropathol Appl Neurobiol 2014;40:177190.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 5. Blümcke I, Kistner I, Clusmann H, et al. Towards a clinico-pathological classification of granule cell dispersion in human mesial temporal lobe epilepsies. Acta Neuropathol 2009;117:535544.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 6. Thom M, Sisodiya SM, Beckett A, et al. Cytoarchitectural abnormalities in hippocampal sclerosis. J Neuropathol Exp Neurol 2002;61:510519.

  • 7. Thom M, Martinian L, Catarino C, et al. Bilateral reorganization of the dentate gyrus in hippocampal sclerosis: a postmortem study. Neurology 2009;73:10331040.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 8. Hudson LP, Munoz DG, Miller L, et al. Amygdaloid sclerosis in temporal lobe epilepsy. Ann Neurol 1993;33:622631.

  • 9. Yilmazer-Hanke DM, Wolf HK, Schramm J, et al. Subregional pathology of the amygdala complex and entorhinal region in surgical specimens from patients with pharmacoresistant temporal lobe epilepsy. J Neuropathol Exp Neurol 2000;59:907920.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 10. Wagner E, Rosati M, Molin J, et al. Hippocampal sclerosis in feline epilepsy. Brain Pathol 2014b;24:607619.

  • 11. Kuwabara T, Hasegawa D, Ogawa F, et al. A familial spontaneous epileptic feline strain: a novel model of idiopathic/genetic epilepsy. Epilepsy Res 2010;92:8588.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 12. Tanaka T, Kaijima M, Yonemasu Y, et al. Spontaneous secondarily generalized seizures induced by a single microinjection of kainic acid into unilateral amygdala in cats. Electroencephalogr Clin Neurophysiol 1985;61:422429.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 13. Majkowski J, Dławichowska E, Sobieszek A. Carbamazepine effects on afterdischarge, memory retrieval, and conditioned avoidance response latency in hippocampally kindled cats. Epilepsia 1994;35:209215.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 14. Shouse MN, Scordato JC, Farber PR. Ontogeny of feline temporal lobe epilepsy in amygdala-kindled kittens: an update. Brain Res 2004;1027:126143.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 15. King JT Jr, LaMotte CC. El mouse as a model of focal epilepsy: a review. Epilepsia 1989;30:257265.

  • 16. Pakozdy A, Sarchahi AA, Leschnik M, et al. Treatment and long-term follow-up of cats with suspected primary epilepsy. J Feline Med Surg 2013;15:267273.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 17. Hasegawa D, Mizoguchi S, Kuwabara T, et al. Electroencephalographic features of familial spontaneous epileptic cats. Epilepsy Res 2014;108:10181025.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 18. Mizoguchi S, Hasegawa D, Kuwabara T, et al. Magnetic resonance volumetry of the hippocampus in familial spontaneous epileptic cats. Epilepsy Res 2014;108:19401944.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 19. Mizoguchi S, Hasegawa D, Hamamoto Y, et al. Interictal diffusion and perfusion magnetic resonance imaging features of familial spontaneous epileptic cats. Am J Vet Res 2017;78:305310.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 20. Lorente de Nó R. Studies on the structure of the cerebral cortex II. Continuation of the study of the ammonic system. J Psychol Neurol 1934;46:113177.

    • Search Google Scholar
    • Export Citation
  • 21. Marcos P, Coveñas R, Narváez JA, et al. Immunohistochemical mapping of enkephalins, NPY, CGRP, and GRP in the cat amygdala. Peptides 1999;20:635644.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 22. Wolf HK, Aliashkevich AF, Blümcke I, et al. Neuronal loss and gliosis of the amygdaloid nucleus in temporal lobe epilepsy. A quantitative analysis of 70 surgical specimens. Acta Neuropathol 1997;93:606610.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 23. Sloviter RS, Kudrimoti HS, Laxer KD, et al. “Tectonic” hippocampal malformations in patients with temporal lobe epilepsy. Epilepsy Res 2004;59:123153.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 24. Baraban SC, Wenzel HJ, Hochman DW, et al. Characterization of heterotopic cell clusters in the hippocampus of rats exposed to methylazoxymethanol in utero. Epilepsy Res 2000;39:87102.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 25. Takai N, Sun XZ, Ando K, et al. Ectopic neurons in the hippocampus may be a cause of learning disability after prenatal exposure to x-rays in rats. J Radiat Res (Tokyo) 2004;45:563569.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 26. Bubier JA, Bennett SM, Sproule TJ, et al. Treatment of BXSB-Yaa mice with IL-21R-Fc fusion protein minimally attenuates systemic lupus erythematosus. Ann N Y Acad Sci 2007;1110:590601.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 27. Rajshankar D, Sima C, Wang Q, et al. Role of PTPα in the destruction of periodontal connective tissues. PLoS One 2013;8:e70659.

  • 28. Martinian L, Catarino CB, Thompson P, et al. Calbindin D28K expression in relation to granule cell dispersion, mossy fibre sprouting and memory impairment in hippocampal sclerosis: a surgical and post mortem series. Epilepsy Res 2012;98:1424.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 29. Wieser HG, ILAE Commission on Neurosurgery of Epilepsy. ILAE Commission report. Mesial temporal lobe epilepsy with hippocampal sclerosis. Epilepsia 2004;45:695714.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 30. Pauli E, Hildebrandt M, Romstöck J, et al. Deficient memory acquisition in temporal lobe epilepsy is predicted by hippocampal granule cell loss. Neurology 2006;67:13831389.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 31. Tsuji A, Amano S, Yokoyama M, et al. Neuronal microdysgenesis and acquired lesions of the hippocampal formation connected with seizure activities in Ihara epileptic rat. Brain Res 2001;901:111.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 32. Sloviter RS, Pedley TA. Subtle hippocampal malformation: importance in febrile seizures and development of epilepsy. Neurology 1998;50:846849.

  • 33. Fatzer R, Gandini G, Jaggy A, et al. Necrosis of hippocampus and piriform lobe in 38 domestic cats with seizures: a retrospective study on clinical and pathologic findings. J Vet Intern Med 2000;14:100104.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 34. Brini E, Gandini G, Crescio I, et al. Necrosis of hippocampus and piriform lobe: clinical and neuropathological findings in two Italian cats. J Feline Med Surg 2004;6:377381.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 35. Schmied O, Scharf G, Hilbe M, et al. Magnetic resonance imaging of feline hippocampal necrosis. Vet Radiol Ultrasound 2008;49:343349.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 36. Pakozdy A, Gruber A, Kneissl S, et al. Complex partial cluster seizures in cats with orofacial involvement. J Feline Med Surg 2011;13:687693.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 37. Klang A, Schmidt P, Kneissl S, et al. IgG and complement deposition and neuronal loss in cats and humans with epilepsy and voltage-gated potassium channel complex antibodies. J Neuropathol Exp Neurol 2014;73:403413.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 38. Klang A, Thaller D, Schmidt P, et al. Bilateral dentate gyrus structural alterations in a cat associated with hippocampal sclerosis and intraventricular meningioma. Vet Pathol 2015;52:11831186.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 39. Fors S, Van Meervenne S, Jeserevics J, et al. Feline hippocampal and piriform lobe necrosis as a consequence of severe cluster seizures in two cats in Finland. Acta Vet Scand 2015;57:41.

    • Crossref
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