Status epilepticus: incidence and latency
There were 108 mice used in this study, 60 DBA mice and 48 A/J mice. Forty-eight animals had status after pilocarpine administration (24 DBA and 26 A/J). and show the number of animals that received each dose of pilocarpine.
Incidence and latency to status epilepticus in DBA and A/J mice
Fig. 1 Incidence and latency to pilocarpine-induced status in DBA and A/J mice. (A) The incidence of status is shown for DBA (white bar) and A/J (black bar) mice. Incidence was defined as the number of animals that entered status out of those that were administered (more ...)
The incidence of status was higher for the A/J strain (; ). Despite the higher incidence of status in A/J mice, the latency to status was usually very long relative to the DBA mice (; ). For some doses, such as the lowest dose (200 mg/kg), few DBA mice entered status, so statistical comparisons were precluded. However, sufficient animals entered status at the 250 mg/kg dose to allow a statistical comparison, and the latency difference was significant (see for data and statistics). For all mice that entered status (all doses pooled), the mean latency was also distinct between strains (DBA: 30±2 min, n=24; A/J: 133±6 min, n=26; Student's t-test, two-tailed, P<0.0000001).
There were acute and chronic differences in mortality that were distinct in the DBA and A/J strains. In the initial 60 min after pilocarpine injections, 18/68 (26%) of mice died during a sudden tonic-clonic seizure (these mice are not included in calculations of incidence or latency in and discussed above, because they did not meet the definition of entering status, i.e. 3 min continuous seizures). The severe tonic-clonic events were almost exclusively in the A/J mice (14/18 mice, or 78%, were A/J mice). The latency from pilocarpine administration to death was not statistically different between the two strains (DBA: 18±3 min, n=4; A/J: 27±5 min, n=14; Student's t-test, two-tailed, P=0.218). For A/J mice, the latency from pilocarpine injection to sudden death was not related to dose (ANOVA for linearity, P=0.302; ), but incidence increased with dose (ANOVA for linearity, P<0.00001). For DBA mice, statistical examination of the relationship between dose and death was not possible due to the low incidence of death.
Although mortality was high in A/J mice in the first hour after pilocarpine administration, the opposite appeared to be true when strains were compared several weeks after status. In other words, mortality was relatively high in DBA mice compared with A/J mice during the chronic period, i.e. weeks after status, and during the time when spontaneous recurrent seizures had developed. Of 15 DBA mice and 14 A/J mice that were followed after they had status, 9 of 15 (60%) DBA mice died in the 3 months after status, but none of the A/J mice died during this period of time (0/14; 0%). This difference was significant (Fisher's exact test, P<0.05). The mean latency to death for DBA mice was 11.6±1.4 weeks after status (range, 5-16 weeks; n=9).
In contrast to acute and chronic differences, there was no evidence of a strain difference in mortality during the 24 h after status: 0/26 A/J mice died, and 3 of 24 DBA mice died (0% vs. 12%, Fisher's exact test, P>0.05). In summary, A/J mice appeared predisposed to acute tonic-clonic seizures ending in death, within 60 min of pilocarpine administration, whereas DBA mice demonstrated mortality in the period of chronic seizures.
Hippocampal electrographic recordingsin vivo
The data described above indicate that A/J mice have a longer latency to status than DBA mice, regardless of dose. However, these data were based on behavioral observation. It is possible that the behavioral signs of status in the A/J mice were misleading, since A/J has not been well-studied with respect to status. Therefore, we considered the possibility that A/J mice had electrographic status earlier than behavioral signs would suggest. To address this possibility, we evaluated electrographic status in a new group of A/J mice. For these experiments, animals were implanted with a hippocampal electrode 1 week before pilocarpine administration (see Experimental Procedures). A 300 mg/kg dose was used to maximize the number of mice that would have status. Concurrent video and electrographic recordings showed that no electrographic manifestations of seizures were detected before, or in the 30 min following atropine administration (; n=3). In all mice that were tested (n=3), EEG seizures did not begin until there were behavioral seizures (). When the behaviors associated with the seizure ceased, so did the electrographic events. When behavioral status began, status began at the electrographic level as well (). Electrographic status was defined by uninterrupted seizure activity in the EEG recording (). The results suggest that the long latency to behavioral status of A/J mice was also the onset of electrographic status, and use of behavioral observation to identify the latency to status was valid for A/J mice.
Fig. 2 EEG recordings from hippocampus of A/J mice. (A) A representative recording from dorsal hippocampus of an awake, behaving A/J mouse, using a bipolar electrode implanted in hippocampus. The trace was recorded 10 min after administration of atropine, 20 (more ...)
Hippocampal changes resulting from status epilepticus in DBA vs. A/J mice
Animals who survived status and had spontaneous seizures were randomly selected for anatomical evaluation at a time when recurrent, spontaneous seizures had begun (at least 4 weeks after status). To minimize potential variability that might be related to the initial dose of pilocarpine, only animals that received 250 mg/kg pilocarpine were used.
To determine whether status led to a different degree of hippocampal pyramidal cell loss in the two strains, animals were perfused at least 4 weeks after status, and sections were evaluated semi-quantitatively using the neuronal marker NeuN. Specifically, DBA mice were perfused 62±20 days after status (range, 30-120 days), and A/J mice were killed 83±25 days after status, (range, 30-150); time to status was not statistically different between strains (Student's t
>0.05). In this analysis, we assumed that the majority of hippocampal pyramidal cell damage had occurred by the time animals were perfused, an assumption based on the evidence that the majority of cell death after status in the rodent occurs in the days after status (Covolan and Mello, 2000
; Wall et al., 2000
; Meldrum, 2002
; Fujikawa, 2005
), and that status, not spontaneous seizures, is primarily responsible for damage (Pitkänen et al., 2002
In the DBA strain, it appeared that a pattern of damage occurred that was typical of Ammon's horn sclerosis, because most of the CA1 and CA3 pyramidal cell layers were devoid of NeuN immunoreactivity, but granule cells and area CA2 appeared to be spared (). In contrast, there was greater preservation of the pyramidal cell layers in the A/J mouse (). The loss of NeuN reflected loss of neurons, rather than a loss of NeuN immunoreactivity, because it was confirmed by Cresyl Violet staining (data not shown).
Fig. 3 NeuN immunoreactivity in DBA and A/J mice. (A) A coronal section through the dorsal hippocampus from an A/J mouse that was injected with saline instead of pilocarpine illustrates the normal neuronal distribution in mouse hippocampus. Densely packed neurons (more ...)
As shown in , there were large sections of the CA3 pyramidal cell layer of DBA mice that were lost. This was present in all DBA mice examined (n=7/7; 100%), but not in any of the A/J mice (n=0/5, 0%; Mann-Whitney U test, P<0.05). For these evaluations, at least three sections, chosen from different anterior-posterior levels of the dorsal hippocampus, were evaluated for all animals, and results from the three sections were the same. All subfields were examined, and the damage in the DBA mouse was only evident in CA3a/b ().
In the same mice that were used to evaluate CA3, CA1 also demonstrated neuronal loss. In four of seven DBA mice there were sections of the cell layer that were devoid of immunoreactivity throughout its entire width (from stratum oriens to stratum radiatum; ). Cresyl Violet staining showed that the loss of immunoreactivity was associated with a loss of neurons. At least three sections were examined per animal, like the examination of CA3. contrast to DBA mice, none of the five A/J mice demonstrated neuronal loss in CA1. The difference between DBA and A/J mice was significant by non-parametric evaluation (DBA, 4 of 7, 57%; A/J, 0/5, 0%; Mann-Whitney U test, P<0.05). The results suggest a distinct pattern of neuronal damage in the DBA strain after status relative to the A/J strain.
It has been shown that adult rats and mice which have had recurrent spontaneous seizures after status demonstrate de novo expression of NPY protein in the axons of granule cells, the mossy fibers (Sperk et al., 1996
; Borges et al., 2003
). However, this does not occur after an individual seizure (Sperk et al., 1996
). Therefore, mossy fiber expression of NPY can be used to confirm that each strain had recurrent seizures prior to being killed. Therefore, we used mossy fiber NPY expression as a tool to confirm that each strain had recurrent seizures. Mossy fiber NPY expression was robust in the hilus and stratum lucidum, as well as the inner molecular layer, indicating de novo expression of NPY and mossy fiber sprouting in both strains (DBA, n
=5 mice; A/J, n
=5; ). None of the saline-treated control mice had mossy fiber NPY expression (n
=7; data not shown). We therefore conclude that both strains developed recurrent seizures (epilepsy), indicating that status leads to spontaneous seizures in both the DBA and A/J mouse. This is significant because some mouse strains do not appear to develop epilepsy after status (McKhann et al., 2003
Fig. 4 Comparison of NPY immunoreactivity in DBA and A/J mice. (A) A tissue section from an A/J mouse that was treated with pilocarpine but had no evidence of seizures, and subsequently was perfused 4 months later to evaluate NPY immunoreactivity in hippocampus. (more ...)
Some qualitative differences are evident in upon comparison of the section from the DBA and A/J mouse. In the A/J mouse, hilar NPY immunoreactivity was greater than in the DBA mouse (). There also appeared to be de novo expression of NPY in some CA1 pyramidal cells (). In the DBA mouse, CA1 stratum radiatum immunoreactivity appeared greater than in the A/J mouse (). However, these differences were not observed in all animals.
Ectopic hilar granule cells
Status epilepticus increases the rate of neurogenesis in the dentate gyrus dramatically, and the effect is consistent across animal models of status (Parent et al., 1997
; for review, see Scharfman, 2004
; Scharfman et al., 2007
). Many of the new neurons enter the hilar region (Parent et al., 1997
; Scharfman et al., 2000
). This also occurs in mice after status (Jung et al., 2004
; Jessberger et al., 2005
). The development of these “ectopic” hilar granule cells depends on a number of the factors, and genetic influence is likely given that the rate of baseline neurogenesis in the normal adult mouse varies with strain (Hayes and Nowakowski, 2002
; Kempermann et al., 2006
). Therefore, we examined the numbers of ectopic granule cells in the hilus in the two strains. Animals were perfused at least 4 weeks after status, a time after status when the population of hilar ectopic granule cells appears stable, based on data from the pilocarpine model in the rat (McCloskey et al., 2006
). As shown in , cell density was greater in DBA mice (DBA: 32,984±8676 cells/mm3
=6; A/J: 6860±1015 cells/mm3
=4; Student's t
-test, two-tailed, P
Fig. 5 Comparison of hilar ectopic granule cells in DBA and A/J mice after status. (A) The numbers of ectopic granule cells in the DBA (white bar) and A/J (black bar) strains after status were compared using Prox1 as a marker of granule cells. Sample size (number (more ...)