Epileptics have chronic recurrent seizures, and epilepsy is sometimes associated with brain dysfunction (eg, memory loss, language impairment, and olfactory dysfunction).1–3 The seizures reportedly induce brain damage, including neuronal death in human epilepsy and epilepsy models in other animals.4–6 Seizure-induced neuronal death is believed to be responsible for the brain dysfunction7 and contributes to the induction of subsequent seizures.8,9 However, the mechanism of seizure-induced neuronal death remains unknown, and an appropriate treatment for prevention of seizure-induced neuronal death has not been identified.
Seizures can induce angiogenesis and microglial activation, which are believed to be related to neuronal death.10–12 Angiogenesis is associated with an increase in local blood flow and is purportedly neuroprotective.13 However, VEGF, which is an important angiogenic factor, has neurodegenerative effects such as induction of neuroinflammation as produced by activated glial cells and breakdown of the blood-brain barrier.10,14 Activated microglia are a major source of proinflammatory cytokines, such as TNF-α and IL-6.15 These cytokines are associated with neuronal damage in several neurologic disorders, including epilepsy, Alzheimer's disease, and amyotrophic lateral sclerosis.16–18
In a colony of Shetland Sheepdogs with familial epilepsy, all dogs had abnormal EEG activity, but some dogs had seizures and others did not. The purpose of the study reported here was to examine whether angiogenesis and microglial activation were related to seizure-induced neuronal death in the cerebral cortex of these dogs.
Materials and Methods
Animals—Ten epileptic Shetland Sheepdogs from the same family and 4 age-matched unrelated Shetland Sheepdogs were used in this study. The related dogs were classified into 2 groups: 6 dogs with a history of recurrent seizures (age range, 20 months to 5 years) and 4 dogs without seizure history (age range, 21 months to 4.4 years). The 4 unrelated dogs, which were used as a control group, ranged in age from 2.3 to 4.6 years and were privately owned dogs that had died from nonneurologic disorders such as tumors or chronic pneumonia. A conventional vaccination schedule had been followed, and a monthly heartworm preventive treatment had been administered. Appropriate veterinary care had been provided when a generalized seizure occurred. For the study, dogs with familial epilepsy were euthanized by administration of pentobarbital. This study was performed with the approval of the Animal Research Committee, in accordance with the guidelines for animal experimentation of the Faculty of Agriculture, Tottori University.
Histologic and immunohistochemical evaluation—A complete postmortem examination was performed on all the dogs, and systemic organs (ie, brain, liver, spleen, kidneys, heart, lungs, and alimentary tract) were fixed in neutral phosphate–buffered 10% formalin. Cerebral tissues were sectioned coronally in 4-mm intervals from the frontal lobe to the occipital lobe. The tissues were then routinely embedded in paraffin and sectioned at 4 μm for H&E staining and immunohistochemical analysis.
For immunohistochemical testing, labeled streptavidin-biotin and double-label immunofluorescence methods were used with primary antibodiesa–d (Appendix). After deparaffinization, tissue sections were transferred into citric acid buffer and boiled for 20 minutes at 98°C in a microwave processore for antigen retrieval according to a standard microwave treatment protocol. For the labeled streptavidin-biotin method, endogenous peroxidase activity was blocked with 3% hydrogen peroxide for 15 minutes and sections were subsequently preincubated with 10% normal goat serum for 5 minutes in the microwave processor. Then, the sections were incubated with a primary antibody in appropriate treatment for each antibody. Sections were sequentially incubated with biotinylated goat anti-rabbit IgGa (diluted 1:400) or biotinylated goat anti-mouse IgGa (diluted 1:400) as a second antibody for 5 minutes in the microwave processor and then incubated with peroxidase-labeled streptavidin complexa for 5 minutes in the microwave processor. The reaction was revealed through application of 3,3′-diaminobenzidine tetrahydrochloride.a Sections were counterstained lightly with Mayer hematoxylin.
For the double-label immunofluorescence method, fluorescent green dyef–conjugated goat anti-rabbit IgGg (diluted 1:100) and fluorescent red dyeh–conjugated donkey anti-mouse IgGg (diluted 1:100) were used as second antibodies. After preincubation with 10% untreated goat serum for 5 minutes in the microwave processor, sections were incubated with rabbit anti–Iba-1 or rabbit anti-GFAP primary antibody and then incubated with fluorescent green dyef–conjugated goat anti-rabbit IgG as secondary antibody for 1 hour at room temperature (approx 20°C). Next, sections were incubated with an anti-mouse primary antibody and then incubated with fluorescent red dyeh–conjugated donkey anti-mouse IgG as secondary antibody for 1 hour at room temperature. Sections were mounted in plain 80% Tris-buffered glycerol. Histochemical evaluation was performed with a confocal imaging system.i Nonspecific staining was confirmed by replacing the primary antibody with PBS solution.
Statistical analysis—Statistical analyses were performed to compare the severity of neuronal death, the number of microvessels, and the number of IL-6–positive cells in tissue specimens from the cingulate cortex of all dogs. For analysis of the severity of neuronal death, an H&E-stained section obtained at the coronal level of corpus mamillare was used and 5 fields of the cingulate cortex and cortex around the sulci (there were no overlaps) were randomly displayed at 200× magnification via light microscopy for each dog with seizures. Healthy neurons (defined as those with a large nucleus with a prominent nuclear body and large cytoplasm with Nissl bodies) and dead neurons (ischemic-like change in neuronal specimens, with shrunken eosinophilic cytoplasm and loss of nuclei) were counted, and the mean percentage of dead neurons was calculated.
For analysis of the number of microvessels, a tissue section immunohistochemically stained with von Willebrand factor antibody (at the coronal level of corpus mamillare) was used, and 10 fields of the cingulate cortex (there were no overlaps) were randomly displayed at 400× magnificationj for each dog (ie, the dogs with seizures, dogs without seizures, and unrelated control dogs). The number of microvessels was counted. The mean number of microvessels was calculated for each dog and each group and compared via the Student t test (normality of data distribution was confirmed with the Kolmogorov-Smirnov test, and equality of variances was checked with the F test).
For analysis of the relationship between the severity of neuronal death and the number of IL-6–positive cells, the severity of neuronal death was defined as the number of neurons with signs of ischemic-like change at 400× magnification. For cerebral tissues from the dogs with seizures, 10 fields of each severity of neuronal death were randomly selected for evaluation at 400× magnification and IL-6–positive cells were counted. The mean number of IL-6–positive cells was calculated for each severity of neuronal death and compared via the Student t test after normality of data distribution was assessed. Summary statistics are reported as mean ± SD.
Results
Animals—The 6 dogs with familial epilepsy in which seizures had been evident had had generalized seizures since they were 1 to 1.5 years of age. The ictal periods ranged from 1 to 4 minutes, and seizure frequency varied from 1 seizure/wk to 1 seizure/6 mo, although the dogs were not watched continuously. Seizures were often observed at night. All dogs with seizures had been continuously monitored by EEG. With onset of seizures, as the seizures became more severe (longer lasting and more frequent), the paroxysmal discharges spread to other regions (parietal and occipital lobe). On the basis of EEG findings, these dogs were considered to have had frontal lobe epilepsy. More details about the clinical history of this family of dogs are described elsewhere.19 In the 4 related dogs lacking clinical epileptic signs, which had also undergone EEG, the paroxysmal discharges (spikes and sharp waves) had been localized in the frontal lobe, particularly the internal area.
Histologic and immunohistochemical evaluation—The 6 dogs with a history of recurrent seizures had histologic evidence of neuronal death (ischemic-like change in neuronal specimens, with shrunken eosinophilic cytoplasm and loss of nuclei) and astrocytosis in the cerebrum (Figure 1).19 Clinically and histologically, no remarkable changes were detected in brain tissues from the 4 related dogs without seizures or the unrelated control dogs.
Representative photomicrographs of tissue specimens from the cerebral cortex (cingulate cortex) of a Shetland Sheepdog with familial epilepsy that had seizures (A) and one from the same family but with no seizures (B). Neurons with ischemic-like change (shrunken eosinophilic cytoplasm and loss of nuclei; arrows) are evident in the specimen from the dog that had seizures, whereas no remarkable changes were observed in the specimen from the other dog. H&E stain; bar = 50 μm.
Citation: American Journal of Veterinary Research 74, 5; 10.2460/ajvr.74.5.763
The neurons with signs of ischemic-like change were isolated to the cingulate cortex (mean ± SD percentage of neurons with ischemic-like change, 84.2 ± 18.9%) and sulci surrounding the cerebral cortex (mean percentage of neurons with ischemic-like change, 34.4 ± 6.8%). No apoptotic bodies were evident. In the related dogs with seizures, microvessels appeared longer and more tortuous than those of the related dogs without seizures in and around the regions of neuronal death (Figure 2). In some instances, the microvessels were surrounded by oval to flat cells, which had positive immunohistochemical results for von Willebrand factor. In tissue specimens from the cingulate cortex of the related dogs with seizures, the mean number of microvessels was significantly greater (82.9 ± 5.9) than in related dogs without seizures (62.5 ± 2.4) or control dogs (61.3 ± 4.6), whereas no significant differences were found between the related dogs without seizures and the control dogs (Figure 3).
Representative photomicrographs of tissue specimens from the cerebral cortex (cingulate cortex) of a Shetland Sheepdog with familial epilepsy that had seizures (A, C, and D) and one from the same family without seizures (B). Microvessels appear longer and more tortuous in the specimen from the dog with seizures (A) than in the specimen from the other dog (B). Oval to flat cells surround the microvessels (C), and these cells have positive immunohistochemical results for the presence of von Willebrand factor (D; arrows) in the dog with seizures. H&E stain (A to C) or labeled streptavidin-biotin for von Willebrand factor (D); bars = 200 μm (A and B), 50 μm (C), or 20 μm (D).
Citation: American Journal of Veterinary Research 74, 5; 10.2460/ajvr.74.5.763
Mean ± SD number of microvessels in the tissue specimens from the cingulate cortex of individual related Shetland Sheepdogs with familial epilepsy with (seizure+) and without (seizure-) seizures and unrelated control dogs of the same breed.
Citation: American Journal of Veterinary Research 74, 5; 10.2460/ajvr.74.5.763
In tissue specimens from the related dogs with seizures, glial cells and neurons in the cingulate cortex and sulci surrounding the cerebral cortex had positive immunohistochemical results for VEGF, whereas VEGF-positive cells were not found in the cerebrum of the related dogs without seizures or control dogs (Figure 4). The double-label immunofluorescence method revealed that the VEGF-positive glial cells consisted of Iba-1–positive microglia and GFAP-positive astrocytes (Figure 5).
Representative photomicrographs of tissues sections from the cerebral cortex (cingulate cortex) of a Shetland Sheepdog with familial epilepsy with seizures (A) and one from the same family without seizures (B). Some glial cells (arrows) and neurons (arrowheads) have positive immunohistochemical results for VEGF in the image for the dog with seizures, whereas no VEGF-positive cells are evident in the image for the other dog. Labeled streptavidin-biotin for VEGF; bar = 50 μm.
Citation: American Journal of Veterinary Research 74, 5; 10.2460/ajvr.74.5.763
Representative photomicrographs of tissue specimens from the cerebral cortex (cingulate cortex) of a Shetland Sheepdog with familial epilepsy that had seizures. A—Fluorescence shows the presence of VEGF (red) and Iba-1 (green) in microglia in separate and merged images. B—Fluorescence shows the presence of VEGF (red) and GFAP (green) in astrocytes in separate and in merged mages. Double-label immunofluorescence; bar = 30 μm.
Citation: American Journal of Veterinary Research 74, 5; 10.2460/ajvr.74.5.763
Greater numbers of microglia with positive results for Iba-1 were evident in the cingulate cortex and sulci surrounding the cerebral cortex in the related dogs with versus without seizures. In those dogs with seizures, Iba-1–positive microglia had a morphologically activated shape (ie, swollen cell bodies and longer processes), which was not observed in tissues from the related dogs without seizures or the control dogs (Figure 6).
Representative photomicrographs of tissue sections from the cerebral cortex (cingulate cortex) of a Shetland Sheepdog with familial epilepsy with seizures (A) and one from the same family without seizures (B). More microglia have positive staining for Iba-1 and swollen cell bodies (arrows) and longer processes in the dog that had seizures than in the other dog. Labeled streptavidin-biotin for Iba-1; bar = 50 μm.
Citation: American Journal of Veterinary Research 74, 5; 10.2460/ajvr.74.5.763
The double-label immunofluorescence method similarly revealed that the activated microglia had positive results for VEGFR-1, TNF-α, and IL-6. The locations of VEGF-positive cells and activated microglia were similar to those of neurons with ischemic-like change. In addition, VEGF-positive cells and activated microglia were also observed in the areas surrounding neurons with ischemic-like change (Figure 7).
Diagrams of the anatomic distribution of neuronal death (black dots) and of activated microglia and cells with results of immunohistochemical testing for VEGF (gray shading) at the coronal level of the corpus mamillare in each of 6 Shetland Sheepdogs with familial epilepsy manifested in seizures. Notice that the distribution of VEGF-positive cells and activated microglia is similar to that of neurons with ischemic-like change. The VEGF-positive cells and activated microglia were also present in the areas surrounding neurons with ischemic-like change.
Citation: American Journal of Veterinary Research 74, 5; 10.2460/ajvr.74.5.763
In the related dogs with seizures, positive staining for IL-6 was evident not only in microglia but also in neurons (except neurons with ischemic-like change) and GFAP-positive astrocytes in the cingulate cortex and sulci surrounding the cerebral cortex (Figure 8). Such positive staining was not identified in tissue specimens from the cerebrum of the other 2 groups of dogs. Statistical analysis revealed that the number of IL-6–positive cells significantly increased with increasing severity of neuronal death. In regions with 5 to 9 neurons with evidence of ischemic-like change, a mean of 21.7 ± 2.3 IL-6–positive cells was observed; in regions with 10 to 19 neurons with ischemic-like change, the mean was 32.8 ± 3.1, and in regions with > 19 neurons with ischemic-like change, the mean was 48.9 ± 2.7. Staining for VEGFR-1 and TNF-α was not detected in GFAP-positive astrocytes.
Representative photomicrographs of tissue specimens from the cerebral cortex (cingulate cortex) in a Shetland Sheepdog with familial epilepsy with seizures (A) and one from the same family without seizures (B). Neurons (black arrowheads) and glial cells (arrows) had positive immunohistochemical results for IL-6, but neurons with ischemic-like change (white arrowheads) lacked IL-6 in the dog with seizures. No IL-6–positive cells were found in the cingulate cortex of the familial dogs without seizures. Labeled streptavidin-biotin for IL-6; bar = 50 μm.
Citation: American Journal of Veterinary Research 74, 5; 10.2460/ajvr.74.5.763
Discussion
In the present study, histologic and immunohistochemical examinations of brain tissue specimens from Shetland Sheepdogs with familial epilepsy manifested in seizures revealed evidence of neuronal death, angiogenesis, and microglial activation that was isolated to the cingulate cortex and sulci surrounding the cerebral cortex. The neuronal death was believed to represent seizure-induced neuronal death because such change was not detected in dogs from the same family without seizures. The seizure-induced neuronal death appeared to be necrosis because no apoptotic cell bodies were detected.
Production of VEGF in neurons and glial cells, long and tortuous microvessels, proliferation of von Willebrand factor–positive vascular endothelial cells, and an increase in the number of microvessels are reportedly related to angiogenesis.10,13 Morphological changes in microglia (swollen cell bodies and long processes) and proinflammatory cytokine production suggest microglial activation.11 In our study, proinflammatory cytokine production was immunohistochemically detected in the cerebral cortex of the dogs with seizures. Immunolabeling of TNF-α was detected in activated microglia. Tumor necrosis factor-α can induce neurotoxic effects by downregulation of astrocytic glutamate transporter 120 (an important transporter for astrocytic glutamate uptake from the synaptic cleft21,22), and the induction of excess glutamate release due to microglial activation in an autocrine manner has been described.23
A previous study19 demonstrated a high concentration of glutamate in the CSF in the cerebrum of dogs with familial epilepsy with versus without seizures, a decrease in positive immunohistochemical results for glutamate transporter 1, and excess glutamate immunopositivity in almost the same distribution as that of neuronal death and activated microglia.24 Glutamate is an excitatory neurotransmitter, and excess glutamate has neurotoxic effects referred to as excitoneurotoxicity.25 Tumor necrosis factor-α, the production of which was induced in the activated microglia in the present study, could cause glutamate metabolism dysfunction, leading to neuronal death.
Immunolabeling of IL-6–positive cells was detected in activated microglia and also in neurons and astrocytes in the cerebral cortex of the Shetland Sheepdogs with seizures. Neuronal hyperexcitability reportedly induces neuronal production of IL-6,26 and IL-6 is believed to have direct and indirect neuroprotective effects against neuronal hyperexcitability.27–29 However, in cerebral cortical tissue specimens from the dogs with seizures, IL-6–positive cells were observed in almost the same distribution as that of neuronal death, and the increase in the number of IL-6–positive cells was related to the severity of neuronal death. The increased number of IL-6–positive cells reflected an increase in the number of IL-6–positive glial cells because neurons with ischemic-like change had no evidence of IL-6.
Overproduction of IL-6 in glial cells has been reported to cause neuronal death.30 In the cerebral cortex of dogs with seizures, production of IL-6 in glial cells may cause local overproduction of IL-6, resulting in neuronal death. Thus, microglial activation with proinflammatory cytokine production was believed to be responsible for the seizure-induced neuronal death in the cerebral cortex of the study dogs with seizures.
Neurons, astrocytes, and microglia in cerebral cortical tissue specimens from dogs with seizures had positive immunohistochemical results for VEGF. Production of VEGF in neurons and astrocytes can be induced by seizures in humans with epilepsy and in models of epilepsy in animals, and VEGF is believed to contribute to angiogenesis for neuroprotection.10,13 However, in the present study, the distribution of the VEGF-positive cells was consistent with that of the activated microglia and the activated microglia were immunohistochemically positive for VEGFR-1. Vascular endothelial growth factor activates microglia via VEGFR-1 and induces an inflammatory response.14,31 In addition, production of TNF-α and IL-6 is induced by VEGF via VEGFR-1 in macrophages and monocytes.32
Circulating monocytes are believed to enter the brain during embryonic development and differentiate into microglia, displaying many cell surface antigens found in macrophages.33 In the cerebral cortex of dogs with seizures, VEGF produced in neurons and astrocytes could activate microglia and induce overproduction of TNF-α and IL-6 in microglia. Moreover, evidence suggests that microglia are not only responsive to but also sources of VEGF.34 The activated microglia in the present study, which were immunohistochemically positive for VEGF, may induce further microglial activation in the cerebral cortex of dogs with seizures.
The angiogenesis and microglial activation detected were believed to be related to seizure-induced neuronal death. However, it could not be determined whether angiogenesis and microglial activation were a cause of seizure-induced neuronal death or sequelae to that death in the study dogs. The VEGF-positive cells and activated microglia were detected not only in the areas of neuronal death but also in the surrounding areas. This pattern suggested that angiogenesis and microglial activation can accelerate seizure-induced neuronal death and expand the lesions. Vascular endothelial growth factor–induced microglial activation with proinflammatory cytokine production is believed to play a critical role in neuronal death. Additional investigation is required into mechanisms of microglial activation and activated microglial damage of neurons in an epileptic brain after a seizure. Such investigations may lead to novel therapeutic approaches for controlling the progression of epilepsy.
ABBREVIATIONS
| EEG | Electroencephalography |
| GFAP | Glial fibrillary acidic protein |
| Iba-1 | Ionized calcium–adapter molecule 1 |
| IL | Interleukin |
| TNF | Tumor necrosis factor |
| VEGF | Vascular endothelial growth factor |
| VEGFR | Vascular endothelial growth factor receptor |
Dako, Glostrup, Denmark.
Wako Chemicals, Richmond, Va.
Santa Cruz Biotech, Santa Cruz, Calif.
Abcam, Tokyo, Japan,
MI-77, Azuma, Tokyo, Japan.
Alexa Fluor 488, Molecular Probes, Eugene, Ore.
Life Technologies, Carlsbad, Calif.
Alexa Fluor 555, Molecular Probes, Eugene, Ore.
AX-70, Olympus Corp, Tokyo, Japan.
BX-51, Olympus Corp, Tokyo, Japan.
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Appendix
Primary antibodies and techniques used for immunohistochemical staining of brain tissues.
| Antibody | Source | Dilution | Incubation |
|---|---|---|---|
| GFAPa | Rabbit polyclonal | 1:100 | 10 minutes in microwave processor |
| Iba-1b | Rabbit polyclonal | 1:400 | 10 minutes in microwave processor |
| IL-6c | Mouse monoclonal | 1:100 | Overnight at 4°C |
| TNF-αc | Mouse monoclonal | 1:300 | Overnight at 4°C |
| VEGFd | Mouse monoclonal | 1:100 | Overnight at 4°C |
| von Willebrand factora | Rabbit polyclonal | 1:100 | 10 minutes in microwave processor |
| VEGFR-1d | Mouse monoclonal | 1:100 | Overnight at 4°C |
