Experimental Models of Status Epilepticus and Neuronal Injury for Evaluation of Therapeutic Interventions

This article describes current experimental models of status epilepticus (SE) and neuronal injury for use in the screening of new therapeutic agents. Epilepsy is a common neurological disorder characterized by recurrent unprovoked seizures. SE is an emergency condition associated with continuous seizures lasting more than 30 min. It causes significant mortality and morbidity. SE can cause devastating damage to the brain leading to cognitive impairment and increased risk of epilepsy. Benzodiazepines are the first-line drugs for the treatment of SE, however, many people exhibit partial or complete resistance due to a breakdown of GABA inhibition. Therefore, new drugs with neuroprotective effects against the SE-induced neuronal injury and degeneration are desirable. Animal models are used to study the pathophysiology of SE and for the discovery of newer anticonvulsants. In SE paradigms, seizures are induced in rodents by chemical agents or by electrical stimulation of brain structures. Electrical stimulation includes perforant path and self-sustaining stimulation models. Pharmacological models include kainic acid, pilocarpine, flurothyl, organophosphates and other convulsants that induce SE in rodents. Neuronal injury occurs within the initial SE episode, and animals exhibit cognitive dysfunction and spontaneous seizures several weeks after this precipitating event. Current SE models have potential applications but have some limitations. In general, the experimental SE model should be analogous to the human seizure state and it should share very similar neuropathological mechanisms. The pilocarpine and diisopropylfluorophosphate models are associated with prolonged, diazepam-insensitive seizures and neurodegeneration and therefore represent paradigms of refractory SE. Novel mechanism-based or clinically relevant models are essential to identify new therapies for SE and neuroprotective interventions

Differential Susceptibility of Interneurons Expressing Neuropeptide Y or Parvalbumin in the Aged Hippocampus to Acute Seizure Activity

Acute seizure (AS) activity in old age has an increased predisposition for evolving into temporal lobe epilepsy (TLE). Furthermore, spontaneous seizures and cognitive dysfunction after AS activity are often intense in the aged population than in young adults. This could be due to an increased vulnerability of inhibitory interneurons in the aged hippocampus to AS activity. We investigated this issue by comparing the survival of hippocampal GABA-ergic interneurons that contain the neuropeptide Y (NPY) or the calcium binding protein parvalbumin (PV) between young adult (5-months old) and aged (22-months old) F344 rats at 12 days after three-hours of AS activity. Graded intraperitoneal injections of the kainic acid (KA) induced AS activity and a diazepam injection at 3 hours after the onset terminated AS-activity. Measurement of interneuron numbers in different hippocampal subfields revealed that NPY+ interneurons were relatively resistant to AS activity in the aged hippocampus in comparison to the young adult hippocampus. Whereas, PV+ interneurons were highly susceptible to AS activity in both age groups. However, as aging alone substantially depleted these populations, the aged hippocampus after three-hours of AS activity exhibited 48% reductions in NPY+ interneurons and 70% reductions in PV+ interneurons, in comparison to the young hippocampus after similar AS activity. Thus, AS activity-induced TLE in old age is associated with far fewer hippocampal NPY+ and PV+ interneuron numbers than AS-induced TLE in the young adult age. This discrepancy likely underlies the severe spontaneous seizures and cognitive dysfunction observed in the aged people after AS activity

Distribution of neuropeptide Y-positive (NPY+) interneurons in different subfields of the hippocampus in an intact aged rat (A1) and an aged rat that underwent three hours of acute seizure (AS) activity (B1).

<p>Figures A2–A4 show magnified views of the dentate gyrus, the CA1 subfield and the CA3 subfield from the figure A1. Figures B2–B4 show magnified views of the dentate gyrus, the CA1 subfield and the CA3 subfield from the figure B1. Note that, three hours of AS activity induces NPY expression in the dentate mossy fibers of the aged rat (B1, B2, and B4). Scale bar, A1 and B1 = 400 µm; A2–A4 and B2–B4 = 100 µm.</p

Distribution of parvalbumin-positive (PV+) interneurons in different subfields of the hippocampus of an intact young adult rat (A1) and a young adult rat that underwent three hours of acute seizure (AS) activity (B1).

<p>Figures A2–A4 show magnified views of the dentate gyrus, the CA1 subfield and the CA3 subfield from the figure A1. Figures B2–B4 show magnified views of the dentate gyrus, the CA1 subfield and the CA3 subfield from the figure B1. Note the paucity of PV+ cell bodies in different hippocampal regions after three hours of AS activity (B2–B4), in comparison to their counterparts in the intact young adult rat (A2–A4). Scale bar, A1 and B1 = 400 µm; A2–A4 and B2–B4 = 100 µm.</p

Distribution of parvalbumin-positive (PV+) interneurons in different subfields of the hippocampus of an intact aged rat (A1) and an aged rat that underwent three hours of acute seizure (AS) activity (B1).

<p>Figures A2–A4 show magnified views of the dentate gyrus, the CA1 subfield and the CA3 subfield from the figure A1. Figures B2–B4 show magnified views of the dentate gyrus, the CA1 subfield and the CA3 subfield from the figure B1. Note the paucity of PV+ soma in different hippocampal regions after three hours of AS activity (B2–B4), in comparison to their counterparts in the intact aged rat (A2–A4). Scale bar, A1 and B1 = 400 µm; A2–A4 and B2–B4 = 100 µm.</p

Comparison of the numbers of neuropeptide Y-positive (NPY+) interneurons in different regions of the hippocampus between the young adult naïve control rats, young adult rats that underwent three hours of acute seizure (AS) activity, aged naïve control rats, and aged rats that underwent three hours of AS activity.

<p>Two-way ANOVA analyses suggested significant main effects of age and AS activity for the dentate gyrus, the CA3 subfield and the entire hippocampus. Additionally, the interaction between age and AS activity was significant in these regions. In the CA1 subfield, there was a significant effect of age but not AS activity; there was also no interaction between age and AS activity (see “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0024493#s2" target="_blank">Results</a>” section for details). Bonferroni post-tests revealed that: (i) the NPY+ interneurons in the dentate gyrus, the CA3 subfield, and the entire hippocampus of aged rats are less vulnerable to AS activity in comparison to their counterparts in young adult rats (A, B); and (ii) the NPY+ interneurons in the CA1 subfield are resistant to AS activity in both aged and young adult animals (C). Additionally note that, the residual numbers of NPY+ interneurons in different hippocampal regions of aged rats after AS activity are significantly lower than numbers in comparable regions of young adult rats that underwent similar AS activity (A–D), which is a consequence of reductions in the numbers of NPY+ interneurons with aging alone (A–D). *, p<0.05; **, p<0.01; ****, p<0.0001.</p

Neurostereology protocol for unbiased quantification of neuronal injury and neurodegeneration

Neuronal injury and neurodegeneration are the hallmark pathologies in a variety of neurological conditions such as epilepsy, stroke, traumatic brain injury, Parkinson’s disease and Alzheimer’s disease. Quantification of absolute neuron and interneuron counts in various brain regions is essential to understand the impact of neurological insults or neurodegenerative disease progression in animal models. However, conventional qualitative scoring-based protocols are superficial and less reliable for use in studies of neuroprotection evaluations. Here we describe an optimized stereology protocol for quantification of neuronal injury and neurodegeneration by unbiased counting of neurons and interneurons. Every 20th section in each series of 20 sections was processed for NeuN(+) total neuron and parvalbumin(+) interneuron immunostaining. The sections that contain the hippocampus were then delineated into five reliably predefined subregions. Each region was separately analyzed with a microscope driven by the stereology software. Regional tissue volume was determined by using the Cavalieri estimator, and cell density and cell number were determined by using the optical disector and optical fractionator. This protocol yielded an estimate of 1.5 million total neurons and 0.05 million PV(+) interneurons within the rat hippocampus. The protocol has greater predictive power for absolute counts as it is based on 3D features rather than 2D images. The total neuron counts were consistent with literature values from sophisticated systems, which are more expensive than our stereology system. This unbiased stereology protocol allows for sensitive, medium-throughput counting of total neurons in any brain region, and thus provides a quantitative tool for studies of neuronal injury and neurodegeneration in a variety of acute brain injury and chronic neurological models