The effects of glycemic control on seizures and seizure-induced excitotoxic cell death
© Schauwecker; licensee BioMed Central Ltd. 2012
Received: 4 May 2012
Accepted: 24 July 2012
Published: 6 August 2012
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© Schauwecker; licensee BioMed Central Ltd. 2012
Received: 4 May 2012
Accepted: 24 July 2012
Published: 6 August 2012
Epilepsy is the most common neurological disorder after stroke, affecting more than 50 million persons worldwide. Metabolic disturbances are often associated with epileptic seizures, but the pathogenesis of this relationship is poorly understood. It is known that seizures result in altered glucose metabolism, the reduction of intracellular energy metabolites such as ATP, ADP and phosphocreatine and the accumulation of metabolic intermediates, such as lactate and adenosine. In particular, it has been suggested that the duration and extent of glucose dysregulation may be a predictor of the pathological outcome of status. However, little is known about neither the effects of glycemic control on brain metabolism nor the effects of managing systemic glucose concentrations in epilepsy.
In this study, we examined glycemic modulation of kainate-induced seizure sensitivity and its neuropathological consequences. To investigate the relationship between glycemic modulation, seizure susceptibility and its neuropathological consequences, C57BL/6 mice (excitotoxin cell death resistant) were subjected to hypoglycemia or hyperglycemia, followed by systemic administration of kainic acid to induce seizures. Glycemic modulation resulted in minimal consequences with regard to seizure severity but increased hippocampal pathology, irrespective of whether mice were hypoglycemic or hyperglycemic prior to kainate administration. Moreover, we found that exogenous administration of glucose following kainic acid seizures significantly reduced the extent of hippocampal pathology in FVB/N mice (excitotoxin cell death susceptible) following systemic administration of kainic acid.
These findings demonstrate that modulation of the glycemic index can modify the outcome of brain injury in the kainate model of seizure induction. Moreover, modulation of the glycemic index through glucose rescue greatly diminishes the extent of seizure-induced cell death following kainate administration. Our data support the hypothesis that deficient insulin signaling may represent a critical contributing factor in the susceptibility to seizure-induced cell death and this may be an important therapeutic target.
Epilepsy is the most prevalent chronic neurologic disorder affecting over 3 million Americans of all ages [1, 2] and is frequently refractory to current medical treatments . Temporal lobe epilepsy (TLE), the most common form of epilepsy, produces a state of chronic neuronal hyperexcitability and hypersynchrony that is manifested as recurrent unprovoked partial seizures . Hippocampal sclerosis, a common feature of TLE [5, 6], is characterized by severe segmental neuronal loss in CA1, CA3, and the hilar region and is accompanied by pronounced astrogliosis . Although the evidence of brain damage in humans, as a result of convulsive status epilepticus (SE), has been difficult to define or quantify, the marked variability in susceptibility to seizure-induced cell damage has been attributed to differences in the underlying pathology, age, and seizure type and duration [8–11]. Regardless, the molecular mechanisms involved in the pathogenesis of hippocampal sclerosis remain highly obscure. Thus, insight into these mechanisms is essential for the development of new neuroprotective drugs as, at present, no effective post-seizure treatment exists to prevent this brain injury.
Many of the pathophysiological consequences of human TLE (e.g. hippocampal sclerosis, mossy fiber sprouting, spontaneous seizures) are faithfully reproduced in the kainic acid (KA) chemoconvulsant rodent model of epilepsy [11–16]. Kainic acid, a potent agonist of the α-amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid/kainate class of glutamate receptors, is a powerful excitant and excitotoxin, which when injected directly into the brain or systemically induces a characterized pattern of persistent seizure activity , activates ionotropic glutamate receptors, and selectively induces excitotoxic cell death in postsynaptic neurons in the CA3 and CA1 hippocampal subfields and within the dentate hilus, while sparing neurons in the dentate granule cell layer [18–21]. Thus, KA administration has been widely used as a model to study excitotoxicity and seizure-related neurologic diseases [17, 22].
While administration of kainic acid to rodents results in acute induction of seizures and subsequent neuronal damage, inbred mouse strains significantly differ in their pattern of hippocampal neurodegeneration in the KA model of TLE [23–29]. Interestingly, the duration or severity of seizure activity in response to KA is not predictive of subsequent hippocampal cell death. Although C57BL/6 (B6) and FVB/N (FVB) mouse strains exhibit comparable seizure activity following systemic administration of kainic acid (KA), C57BL/6 mice show essentially no hippocampal cell death. Those mice susceptible to KA administration show an excitotoxic response similar to what has been described in rats [18, 30–32].
Metabolic disturbances are often associated with epileptic seizures, but the pathogenesis of this relationship is poorly understood. The importance of glucose balance has been identified in studies demonstrating that epileptic seizures can be exacerbated under conditions of hyper- or hypoglycemia [33–35]. Studies of type I and type II diabetic subjects have found that diabetes-related seizures usually improve with control of glycemic status . In particular, the treatment of choice for hyperglycemia-related seizures is glycemic control, and seizures are usually resistant to antiepileptic drugs if blood glucose is not brought under control . At present, the mechanisms underlying glucose regulation and altered neuronal excitability remain incompletely understood . Nevertheless, despite the reported association between blood glucose levels and certain epilepsy syndromes , few studies to date have evaluated the efficacy of controlling blood glucose levels on seizure-induced neuronal injury.
In this study, we used mice that possess strain-specific gene products that can modify vulnerability to excitotoxin-induced cell death . We were interested in identifying the relationship between glycemic index and susceptibility to seizure-induced excitotoxic cell death and establishing whether modulation of glycemic index could modify the susceptibility to epilepsy. As a first step, we wanted to determine if glucose administration following kainate-induced SE could reduce seizure-induced cell death in mice susceptible to excitotoxin-induced cell loss (FVB/N). Secondly, we wanted to determine if modulation of the glycemic index, in models of hypoglycemia or hyperglycemia, could affect seizure susceptibility and its neuropathological consequences following kainate administration in a mouse strain previously found to be resistant to seizure-induced cell death (C57BL/6).
Effect of kainic acid administration on seizure parameters in normoglycemic, hypoglycemic and hyperglycemic mice
Stages 1-4 (% of mice)
Stage 5 (% of mice)
32.8 ± 1.6
69.6 ± 4.7
30.5 ± 1.3
76.3 ± 2.8
30.4 ± 6.4
55.4 ± 14.4
49.6 ± 6.1 a
66.2 ± 4.0
36.5 ± 4.6
93.3 ± 2.9 b
In accordance with previous studies [23, 27], quantitative analysis of subfield group means revealed that mice susceptible to seizure-induced cell death via KA administration (FVB) displayed a reduction of 44% dentate hilar neurons, 64% of CA3 pyramidal neurons, and 49% of CA1 pyramidal neurons seven days after KA administration (F=17.72 ; P<0.001; Figure 2B). In contrast, administration of glucose significantly reduced neuronal damage in FVB mice (F=16.38; P<0.001). Post hoc analyses revealed a dramatic protective effect of glucose following KA administration in area CA3 (p<0.001), area CA1 (p<0.001); and the dentate hilus (p< 0.001), as compared to FVB mice treated with KA alone.
To determine if hypoglycemia could modulate seizure severity, B6 mice were treated with kainic acid and seizure severity was assessed. As shown in Table 1, we found no difference between B6 mice that were hypoglycemic versus vehicle injected mice with regard to seizure sensitivity, as evidenced by no alterations in latency to onset of first severe seizure (P=0.990) or duration of severe seizures (P=0.24). Thus, hypoglycemia was without effect on KA-induced seizure activity.
Following systemic administration of KA to young adult B6 mice, essentially no cell death was observed within the hippocampus or within any other region of the brain, in accordance with previous results [23, 27], and as evidenced by no loss of cresyl violet or NeuN-immunostaining (Figure 3C, panels D,E). In contrast, in B6 mice with insulin-induced hypoglycemia followed by KA-induced SE, we observed significant cell loss, as evidenced by decreased cresyl violet (Figure 3C, panel G) and NeuN-immunostaining in three hippocampal subfields (dentate hilus, area CA3 and area CA1; Figure 3C, panel H).
In particular, quantitative analysis of subfield group means revealed that hypoglycemic B6 mice that underwent KA-induced SE displayed a reduction of nearly 50% of dentate hilar neurons, over 80% of CA3 pyramidal neurons, and nearly 70% of CA1 pyramidal neurons (Figure 3B). Thus, a significant reduction in hippocampal neurons within the dentate hilus (F=64.31; P<0.001), area CA3 (F=150.33; P<0.001) and area CA1 (F=99.70; P<0.001) was observed. In contrast, normoglycemic B6 mice that underwent KA-induced SE displayed no detectable evidence of reduction of neurons in any of the hippocampal subfields after KA administration. These results demonstrate that B6, which are typically excitotoxin cell death resistant, exhibit seizure-induced cell death when they undergo insulin-induced hypoglycemia followed by KA-induced SE.
Previous studies have suggested that glycemic modulation can alter seizure threshold [44, 46]. Thus, in the present study, we used two models of hyperglycemia to assess the effects of glycemic modulation on seizure induction and seizure duration in B6 mice. While we found no qualitative differences in seizure intensity irrespective of the model utilized to induce hyperglycemia (no differences in the percentage of mice achieving status epilepticus, Table 1), we did find differences between the models with regard to latency to onset of stage 4 seizures (Table 1). In particular, we found a significant increase in latency to onset of stage 4 seizures in our model of hyperglycemia as compared to vehicle-injected animals (KA=30.50 ± 1.32 mins vs. Hyperglycemia + KA= 49.60 ± 6.06 mins, P=0.03), but no significant difference in latency when we compared STZ-induced hyperglycemia vs. vehicle alone (P=0.25). As well, we found significant differences in the duration of stage 4 seizures depending on our model of induction of hyperglycemia. While we found no significant differences in seizure duration between hyperglycemic versus vehicle-injected mice (P=0.09), we did find a significant difference in seizure duration when comparing STZ-induced hyperglycemic mice versus controls (KA=76.25 ± 2.83 vs. STZ + KA= 93.25 ± 2.90, P=0.006).
Quantitative analysis of hippocampal subfield group means (Figure 5B) revealed a significant reduction in neuronal cell loss within the dentate hilus (F=23.28; P<0.001), area CA3 (F=64.73; P<0.001) and area CA1 (F=112.57; P<0.001) of diabetic hyperglycemic mice that underwent KA-induced SE. Thus, we found that compared with non-diabetic mice, diabetic mice lost more hippocampal neurons during the acute stage after status epilepticus. In contrast, we observed no significant cell loss in B6 mice that were injected with vehicle prior to KA-induced SE. These results demonstrate that B6, which are typically excitotoxin cell death resistant, exhibit seizure-induced cell death when they undergo diabetes-induced hyperglycemia followed by KA-induced SE.
Quantitative analyses of hippocampal neuron numbers showed a dramatic and significant reduction on average of 90% of dentate hilar neurons (F=781.34; P<0.001), CA3 pyramidal neurons (F=542.49; P<0.001), and CA1 pyramidal neurons (F=567.89; P<0.001) 7 days after KA administration as compared with normoglycemic mice (Figure 6B). These results indicate that glycemic modulation can induce differential vulnerability of neurons in the hippocampus.
The effects of glycemic modulation on excitotoxic cell death and seizure susceptibility are diverse and complex. It has previously been established that limited energy availability compromises hippocampal neuronal viability during status epilepticus [51–53]. As seizure-induced excitotoxic cell death is likely due to excessive glutamate release and excessive cytosolic calcium , these can be worsened by energy failure. In particular, intracellular sequestering of Ca2+ and the concomitant transmembrane extrusion of Ca2+ are directly or indirectly ATP-dependent , and can result in impaired ATP production , the release of reactive oxygen species , and the subsequent release of proteins involved in the cell death cascade [58–61]. As a result, many of the steps mediating seizure-induced excitotoxic cell death are sensitive to energy availability. In the present study, we examined the effects of glycemic modulation on excitotoxic cell death and seizure susceptibility following systemic administration of the chemoconvulsant, kainic acid to inbred strains of mice. We demonstrated that injection of glucose following KA-induced SE was profoundly neuroprotective against seizure-induced neuronal damage. As well, we also found that compared with normoglycemic mice, (1) hyperglycemic were less seizure sensitive, (2) diabetic hyperglycemic mice were more seizure sensitive, and (3) hypo-, hyper- or STZ-hyperglycemic mice showed increased susceptibility to seizure-induced cell death after status epilepticus.
In order to assess the putative ‘neuroprotective’ effects of controlling glycemic status in mice that underwent KA-induced SE, we administered glucose 3 hours following KA-induced status epilepticus and then gave two additional injections 24 and 48 hours following the initial glucose injection. We report that FVB mice are hypoglycemic following KA-induced SE and that glucose treatment reverses the hypoglycemic status as well as provides protection against seizure-induced cell death. Our results are in agreement with previous clinical and experimental studies on ischemic brain injury . Preclinical data from animal models indicates that insulin may reduce damage in both global and focal ischemia [62, 63] and in transient global ischemia, insulin has a direct neuroprotective effect on CNS parenchyma . Moreover, Nagamizo et al.  demonstrated that a relatively small dose of preischemic insulin protects against ischemic spinal cord injury, and that the protective effect was cancelled by a concomitant glucose infusion. The idea of brain protection by insulin is not new; however, the mechanisms underlying glucose levels and altered neuronal excitability remain incompletely understood.
As previous studies have established that there is a biphasic dependence of seizure susceptibility on blood glucose concentrations, we examined several seizure parameters (seizure intensity, seizure latency and seizure duration) in hypoglycemic and hyperglycemic B6 mice. While we found no difference between B6 mice that were hypoglycemic versus vehicle-injected mice with regard to seizure sensitivity, latency to onset of first severe seizure or duration of severe seizures, we did observe that hyperglycemia can modulate seizure susceptibility. In particular, while we found no qualitative differences in seizure intensity irrespective of the model utilized to induce hyperglycemia, we did find differences between the models with regard to latency and duration.
Interestingly, hyperglycemic mice that underwent KA-induced SE demonstrated a significant increase in the latency to onset of severe seizures, suggesting a reduction in seizure severity. In contrast, STZ mice displayed a significant increase in seizure duration following KA-induced SE, indicative of an increase in seizure sensitivity. Thus, depending on the model of hyperglycemia utilized, B6 mice appeared to be either less seizure sensitive (hyperglycemia) or more seizure sensitive (STZ-induced hyperglycemia). While no studies to date have compared seizure susceptibility differences based on the model of hyperglycemia, previous studies have found that STZ-induced diabetic rats that underwent lithium-pilocarpine induced SE had a higher seizure susceptibility than normoglycemic rats .
It is somewhat surprising that we saw differential effects on seizure susceptibility depending on the model of hyperglycemia utilized in our studies, as others have demonstrated that seizure susceptibility has been shown to increase with incremental blood glucose, either during experimental diabetes or acute hyperglycemia [38, 67]. One potential reason for the discrepancy between our models of hyperglycemia could be the result that we compared a bolus injection of glucose to create a condition of nonketotic hyperglycemia independent of diabetes versus the STZ-induced model of type 1 diabetes (chemical ablation of the pancreatic ß cells; ). The underlying mechanism for the difference in seizure susceptibility between the high glucose model and STZ model is uncertain, although the high glucose model has been described as an acute hyperglycemia model, while the STZ model is described as a chronic or sustained hyperglycemia model.
Previous studies in our laboratory have demonstrated robust strain differences with respect to susceptibility to excitatory amino acid-induced cell death [26–28]. Although C57BL/6 (B6) and FVB/N (FVB) mouse strains exhibit comparable seizure activity following systemic administration of kainic acid, FVB mice have been reported to be vulnerable to excitotoxic insults, while B6 mice are resistant to excitotoxic cell death. While the molecular and cellular events responsible for the selective vulnerability of hippocampal neurons to kainic acid are not yet fully understood, excitotoxicity is thought to be triggered by the activation of ionotropic glutamate receptors resulting in calcium dysregulation [22, 54, 69], oxidative stress and mitochondrial dysfunction, and the initiation of signaling cascades within susceptible neurons resulting in cell death [70, 71].
Our results demonstrate that hypoglycemia induced a phenotypic switch in B6 mice, previously characterized as resistant to seizure-induced excitotoxic cell death. In particular, B6 (excitotoxin cell death resistant) mice with insulin-induced hypoglycemia following by KA-induced SE exhibited greater seizure-induced cell death. We are not aware of any studies that compared the effects of hypoglycemia on status epilepticus associated injury. However, our results suggest that hypoglycemia aggravates excitotoxic neuronal death in a nearly excitotoxin-resistant brain.
The mechanism of hypoglycemia-induced injury is thought to be the result of several contributing factors acting downstream of glucose deprivation, and not just a loss of energy supply (e.g. glucose) from neurons . One of these contributing factors includes the sustained activation of glutamate receptors , as well as increased mitochondrial membrane permeability . Differences in the number and function of neurotransmitter receptors, molecular and biochemical pathways of energy metabolism and/or susceptibility to respiratory depression may also be responsible for the increased vulnerability to excitotoxic cell death. At present, while we cannot explain why induction of hypoglycemia prior to KA-induced SE renders mice previously characterized as excitotoxin cell death resistant to become susceptible; it is possible that excessive glutamate activation (via acute hypoglycemia combined with stimulation of ionotropic glutamate receptors via kainate administration) drives glutamate release to excessive levels that cannot be resolved when only one insult is presented. Additional studies must clarify the specific cellular mechanisms that promote neurotoxicity in C57BL/6 mice.
In the present study, we found that STZ-induced and glucose-induced hyperglycemia exacerbated the neuropathological consequences of KA-induced SE in excitotoxin-resistant mice. Thus, both acute and chronic hyperglycemia produced the same outcome of increased susceptibility to seizure-induced excitotoxic cell death. Our results demonstrating that both models of hyperglycemia rendered hippocampal neurons more vulnerable to KA-induced excitotoxic cell death are in agreement with previous clinical and animal studies. In particular, previous studies have demonstrated that status epilepticus and its associated neuropathological consequences is not an uncommon complication associated with diabetic hyperglycemia [33, 35]. Diabetes has been suggested to exacerbate status epilepticus-induced brain damage, result in poor recovery following status, and even increase mortality [38, 75, 76]. Moreover, animal studies have demonstrated that high glucose concentrations are associated with an increased susceptibility to seizures and augmented brain damage in the pilocarpine model of temporal lobe epilepsy . Hyperglycemia is known to modify many proteins important for cell survival by advanced glycation, inducing some death-related proteins like high mobility group box 1, which triggers the expression of pro-inflammatory mediators, or downregulation of cytoskeletal proteins that support neuronal cell survival [77, 78]. Thus, the prolonged effects of hyperglycemia may be the result of an additive effect of toxicity on the brain.
In summary, we found that glycemic control could rescue hippocampal cells from seizure-induced excitotoxic cell death in an excitotoxin-susceptible mouse strain, FVB. As well, the results presented here illustrate that hyper- or hypoglycemia additively increased the extent of seizure-induced cell death in an excitotoxin-resistant mouse strain, B6. The ability of glucose dysregulation to elicit a phenotypic switch from excitotoxin resistant to susceptible after kainate administration implicates glucose dysfunction as a key event in the pathogenesis of seizure-induced excitotoxic cell death. While the specific pathophysiological mechanisms underlying this relationship remain unclear, our data support the hypothesis that deficient insulin signaling may represent a critical contributing factor in the susceptibility to seizure-induced cell death and this may be an important therapeutic target. An understanding of the interplay between glucose regulation and excitotoxic neurodegeneration will have important consequences, not only in the context of epilepsy, but for hypoxia, stroke, and other related pathologies.
All experiments were conducted in accordance to the guidelines set forth by the National Institutes of Health. All procedures were approved by the University of Southern California Institutional Animal Care and Use Committee. Animals were housed under controlled conditions (12 hour light/12 hour dark), and food and water were provided to the mice ad libitum.
Four separate cohorts of mice were used for these studies. Six to eight-week old FVB/N male mice (Jackson Laboratories, Bar Harbor, ME), housed in groups of 4–5 per cage, were used for the KA + exogenous glucose experiments. Three separate cohorts of six to eight-week old C57BL/6 (Jackson Laboratories, Bar Harbor, ME), housed in groups of 4–5 per cage, were used for the hypoglycemia + KA, hyperglycemia + KA, and Streptozotocin (STZ) + KA experiments. All of the procedures used in these experiments were in accordance with the NIH Guide and approved by the USC Animal Care and Use Committee. All efforts were made to minimize the number and suffering of any animals used in these experiments.
On the experimental day, mice were weighed and the baseline blood glucose was measured. Mice were injected i.p. with 1 I.U./kg of regular human insulin (Humalin, Eli Lilly, Indianopolis, IN), freshly dissolved in normal saline. Blood glucose from blood was measured prior to insulin administration (basal glucose concentration), and at 2 additional timepoints. Blood glucose was measured 30 minutes following insulin administration, prior to kainate administration and 3 hours following kainate administration. Mixed peripheral blood (arterial, venous, and capillary) samples were obtained after snipping the tip of the tail. A droplet of blood (~2 μl) was collected on the test strip and evaluated using a One-Touch Glucosemeter (LifeScan, Millipitas, CA). As the sample size of the blood droplet was minimal, there was no adverse effect to the well being of the mouse and only a single cut of the tip of the tail was needed to provide all samples. Blood glucose levels of <5.1 mM were defined as hypoglycemic.
One group of mice from each strain was fasted overnight and intraperitoneally injected with 1 ml of a 20% glucose solution 30 minutes prior to KA-induced SE to create a condition of nonketotic hyperglycemia independent of diabetes . Blood glucose from blood was measured prior to glucose administration (basal glucose concentration) and 3 hours following kainate administration.
A second group of mice was fasted overnight and administered 200 mg/kg, i.p. streptozotocin (STZ; Sigma Aldrich, St. Louis, MO) in 100 mM citrate buffer (pH 4.5), an antibiotic that destroys the insulin-secreting ß cells of the pancreas [47, 48] and has previously been used to induce chronic hypoinsulinemia in rodents [49, 50]. Controls received citrate buffer alone. Both sets of mice underwent kainate-induced status epilepticus three weeks following drug administration. Blood glucose was measured prior to STZ administration (basal glucose concentration), and monitored weekly following STZ administration (for a total of 3 weeks) in peripheral blood obtained after snipping the tail. A droplet of blood (~2 μl) was collected on the test strip and evaluated using a One-Touch Glucosemeter (LifeScan, Millipitas, CA). Mice with blood glucose values > 250 mg/dl were included in the STZ group.
Sustained seizures (status epilepticus) were induced in animals by the administration of kainic acid (KA), a potent agonist of the AMPA/KA class of glutamate receptors. KA was dissolved in isotonic saline (pH 7.4) and administered subcutaneously to adult mice at a dose of 20 mg/kg (Nanocs, New York, NY). Following KA administration, mice were monitored continuously for 4 h for the onset of locomotor activity and behavioral manifestations of limbic seizure episodes, as described previously [23–26, 28]. Status epilepticus was defined as continuous behavioral seizure activity lasting at least 1 hour or a series of intermittent seizures without restoration of normal behavioral patterns between successive seizures. Mice were scored for seizure activity using a previously defined six-point seizure scoring scale  that was adapted from a five-point scale for rats .
Seizure stages were defined as follows: Stage 1, immobility; Stage 2, forelimb and/or tail extension, rigid posture; Stage 3, repetitive movements, head bobbing; Stage 4, rearing and falling; Stage 5, continuous rearing and falling; and Stage 6, severe tonic-clonic seizures. Only those mice exhibiting at least 45 min of continuous stage 4/5 seizures were included in this study, as previous studies have suggested that there is a direct relationship between the generation of epileptiform activity and the extent of damage in hippocampal subfields [18, 32, 80]. Seizure parameters monitored included latency of convulsions and duration of severe (Stage 4/5) seizure activity. All experiments were approved by the Institutional Animal Care and Use Committee (IACUC) of the University of Southern California and conducted in accordance with its guidelines. Every effort was made to minimize animal suffering and to minimize the number of animals utilized in order to produce reliable scientific data.
Administration of 1 ml of 20 mg/ml glucose (intraperitoneal) was initiated 3 hours following KA-induced status epilepticus and two additional injections were given 24 and 48 hours following the initial glucose injection. Blood glucose from blood was measured prior to glucose administration and 30 minutes following each glucose injection.
In order to evaluate the severity of kainate-induced excitotoxic brain damage, brains from each strain of mice were processed for light microscopic histopathologic evaluation according to previously published methods . Briefly, 7 days after seizure induction by kainate, mice were anesthetized with Avertin and transcardially perfused with 4% paraformaldehyde in 0.1M phosphate buffer (pH 7.4). Brains were removed and post-fixed overnight, followed by cryoprotection in 30% sucrose for at least 12–18 h. Horizontal (40 μm) frozen sections were cut on a sliding microtome and collected as free-floating sections in 0.1M phosphate buffer (pH 7.4) until processed for light microscopic histology. Every sixth section (~240 μm) was processed for cresyl violet staining to assess cell loss and morphology.
Immunofluorescence was performed on an additional series of sections (every sixth section; ~240 μm) to detect those neurons that survived 7 days following kainate-induced SE. For immunofluorescent labeling, sections were washed with 0.1M phosphate buffer (pH 7.4) and blocked with 5% normal serum and 0.1% Triton X-100 in 0.1M phosphate buffer (pH 7.4). Next, sections were incubated overnight with a neuronal marker against NeuN (monoclonal from mouse; Millipore, Billarica, MA; 1:500) at 4°C. After several washes, sections were incubated with a secondary antibody from mouse conjugated with Cy2 (1:200; Jackson ImmunoResearch, West Grove, PA) for 2 h at room temperature. After rinsing, sections were mounted and coverslipped with ProLong anti-fade mounting medium (Molecular Probes, Eugene, OR). For labeling, omission of the primary antibody served as negative control. Labeling for NeuN was viewed under an Olympus BX51 fluorescence microscope (Olympus, New York, NY).
Subsequently, to determine the susceptibility of individual hippocampal subfields to neurotoxic insult, we counted neurons in Nissl-stained sections. Quantitative analysis of hippocampal cell loss was performed by an observer blinded to the strain groups using unbiased stereological methods on cresyl violet-stained sections according to previously published protocols [23, 27, 28]. The Nissl-stained neurons in area CA3, area CA1, the dentate hilus, and the dentate gyrus were counted in both the right and left hippocampus and counting was initiated within the ventral hippocampus at the first point where hippocampal subfields could be easily identified. This level corresponded to horizontal section 54, based on the atlas of Sidman et al. . Hippocampal subfields were based on Franklin and Paxinos  classification and discrimination between the CA3 and the dentate hilus region was based on morphological features and locations of the cells [83, 84]. Specifically, for dentate hilar cell counts, the hilus was operationally defined as the region bordered by the supra- and infrapyramidal granule cell layers and excluding the densely packed pyramidal neurons of area CA3.
Neuron counts were made in all subfields and the numbers for each side were averaged into single values for each animal. Surviving cells were counted only if they were contained within the pyramidal cell layer, dentate hilus or dentate gyrus, possessed a visible nucleus and characteristic neuronal morphology and had a cell body larger than 10 μm. Six square counting frames (200 X 200 μm) were randomly placed in the pyramidal layer of fields CA1 and CA3 or in the dentate gyrus in 4–5 regularly spaced horizontal sections from each animal. Two square counting frames were randomly placed in the dentate hilus in 4–5 regularly spaced horizontal sections from each animal. Neuronal nuclei were evaluated at three different focal planes and only those in the focal plane were counted with a 40X objective and considered as a counting unit. Stereological analysis was performed with the aid of ImagePro Plus 4.5 software (Media Cybernetics, Silver Spring, MD) and a motorized Z-stage (Optiscan; Prior Scientific, Fairfax, VA). Final cell counts were expressed as the percentage of cells as compared to intact mice. Results were assessed statistically by one-way analysis of variance (ANOVA) using the computer program, SigmaStat (Jandel Scientific, San Rafael, CA), and intergroup differences were analyzed by Newman-Keuls post hoc test.
Analysis of variance
Central nervous system
Kainate-induced status epilepticus
Temporal lobe epilepsy.
This work was supported by NINDS RO1 NS380696. The author wishes to thank Margaret Kornacki for technical assistance.
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