Brain Advance Access originally published online on March 9, 2005
Brain 2005 128(6):1344-1357; doi:10.1093/brain/awh475
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Quantitative post-mortem study of the hippocampus in chronic epilepsy: seizures do not inevitably cause neuronal loss
Department of Clinical and Experimental Epilepsy, Institute of Neurology, University College, London, UK
Correspondence to: M. Thom, Division of Neuropathology, National Hospital for Neurology and Neurosurgery, Queen Square, London WC1N 3BG, UK E-mail: M.Thom{at}ion.ucl.ac.uk
Received October 15, 2004. Revised December 16, 2004. Accepted February 10, 2005.
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Summary |
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Hippocampal sclerosis describes a pattern of neuronal loss and gliosis involving the medial temporal structures most often encountered in patients with epilepsy. It is still a matter for debate as to whether this lesion is acquired during the course of the patient's seizure history or is present at the outset. Early febrile seizures, episodes of status epilepticus as well as repetitive brief seizures may all contribute to the evolution of hippocampal sclerosis. In addition, genetic factors and developmental abnormalities of the hippocampus may both increase vulnerability to seizures and hippocampal injury. Recent human studies have addressed neuropathological changes in young adults and children undergoing surgery for refractory seizures with hippocampal sclerosis. Post-mortem examination, however, provides the opportunity to evaluate the effect of a lifetime of seizures on both left and right hippocampi, and the presence of any co-existing malformation. Post-mortem stereological analysis of 28 patients with poorly controlled seizures has confirmed a subgroup with absence of significant hippocampal neuronal loss despite decades of generalized seizures, including status epilepticus. The presence of granule cell dispersion correlated to the severity of hippocampal neuronal loss. Furthermore, in patients with confirmed hippocampal sclerosis at post-mortem examination, stereological assessment of the neocortex failed to confirm significant white matter neuronal heterotopia that might indicate an underlying developmental abnormality. In conclusion, seizures do not invariably lead to hippocampal injury and white matter heterotopia is not invariably associated with hippocampal sclerosis.
Key Words: hippocampal sclerosis; epilepsy; post-mortem; quantitation; development
Abbreviations: GTCS = generalized tonic–clonic seizure; HS = hippocampal sclerosis; IPI = initial precipitating injury; LFB = Luxol Fast Blue; MTLE = mesial temporal lobe epilepsy; PHG = parahippocampus gyrus; TLE = temporal lobe epilepsy
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Introduction |
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Hippocampal sclerosis is the commonest lesional abnormality identified in temporal lobe epilepsy (Bruton, 1988
; Blumcke, 2002). It is considered to be acquired, but its exact aetiology is uncertain. Seizures are known to damage the hippocampus, particularly prolonged seizures and status epilepticus (Men et al., 2000; Salmenpera et al., 2000; Pitkanen et al., 2002; Scott et al., 2002). The timing of the first insult may be critical to the development of hippocampal sclerosis. Prolonged febrile seizures early in life (Lewis, 1999; Scott et al., 2003; Baulac et al., 2004; Cendes, 2004) or other forms of cerebral injury may initiate the process of irreversible and progressive hippocampal damage. There is also evidence from some (Briellmann et al., 2001, 2002) but not all (Liu et al., 2002; Holtkamp et al., 2004) longitudinal MRI studies that chronic repetitive seizures result in progressive hippocampal changes suggesting cumulative damage. As neither status, prolonged febrile seizures or repetitive seizures inevitably lead to hippocampal sclerosis, there are likely to be other factors, including a contributing genetic susceptibility to hippocampal sclerosis. There is also considerable speculation and some accumulating evidence that developmental hippocampal abnormalities increase susceptibility to seizures and neuronal loss, and act as a template for subsequent hippocampal sclerosis (Baulac et al., 1998; Fernandez et al., 1998; Grunewald et al., 2001; Blumcke et al., 2002). Most neuropathological studies of hippocampal sclerosis have been carried out on surgical resections of selected patients with MRI-proven asymmetrical hippocampal damage. We have had the opportunity to study a large collection of post-mortem tissues from patients with long, well-documented histories of refractory seizures. This has allowed us to address relationships between the effect of lifetime seizures on the hippocampus of both hemispheres and to assess for the presence of temporal lobe developmental lesions.
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Methods |
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Case and tissue selection
Post-mortem cases were retrieved from the archives of the Division of Neuropathology at the National Hospital for Neurology and Neurosurgery, Queen Square, London, UK, between 1984 and 2002. All patients had been long-term residents at the Chalfont Centre for Epilepsy (National Society for Epilepsy), Gerrards Cross, Buckinghamshire, UK. This study was approved by the Joint Research Ethics Committee of the National Hospital for Neurology and Neurosurgery; consent for post-mortem examination and retention of brain tissue for research purposes was granted.
Eight cases were selected on the basis of evidence of classical hippocampal sclerosis (HS) involving one or both hemispheres with the characteristic patterns of neuronal loss and gliosis involving CA1 and CA4 subfields on routine Nissl and GFAP stained sections (HS group: mean age 78 years; age range 59–85 years). A second group of nine cases with apparent absence of hippocampal sclerosis on qualitative examination alone was also selected (non-HS group: mean age 64 years; range 46–85 years). A further control group selected from the pathology archives of seven patients without a history of epilepsy or other neurological disease (mean age 60 years; range 34–85 years) was also studied.
From each case, paraffin-embedded tissue blocks were selected from right and left hippocampus, at the level of the lateral geniculate nucleus. Further sections were cut at 25 µm thickness and stained with Luxol Fast Blue (LFB)/cresyl violet or cresyl violet alone. All hippocampal specimens were identically processed to minimize the effect of differential shrinkage on neuronal density measurements.
For the study of temporal lobe malformations, a further four cases without hippocampal sclerosis and seven cases with hippocampal sclerosis were selected from the pathology archives. Tissue blocks were selected and sections taken as above. The clinical notes from all patients were retrieved and reviewed, the neuropathological and clinical data are presented in Tables 1–3. In particular, information was sought and noted regarding the type of seizures, the age of onset, if there was an initial precipitating injury (IPI) for the seizures (including prolonged or complex febrile convulsion, cerebral trauma or other cerebral injury), the duration, frequency and minimum total number of lifetime seizures (each event having been recorded throughout their admission) and any episodes of status epilepticus.
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Quantitative analysis of hippocampal neurons
Neuronal cell counts were performed using a Histometrix stereological analysis system (Kinetic Imaging, Nottingham, UK) incorporating a Zeiss Axioskop microscope. In each LFB/Nissl stained section, CA1, CA2, CA3, CA4 and subicular regions were identified and outlined at a magnification of 2.5x objective. The identification of each area was made according to known architectonic and topographic data (Duvernoy, 1988
) (Fig. 1A) and the whole of the pyramidal cell layer in each subfield was outlined. Within each delineated region, neurons were counted at a magnification of 63x (aperture 1.4) using the optical dissector method and a counting box of area 160 µm2 x 10 µm deep. Only principal neurons (i.e. those with large pyramidal or multipolar shape) containing a prominent nucleolus and Nissl positive cytoplasm were included in the analysis and the neuronal densities were calculated per mm3. Small cells, which may represent either glia or interneurons, were not included as their true lineage could not be confirmed without immunohistochemistry. In a pilot study of four cases, a systematic random sample of 10% of the region gave results with minimal sampling bias, and this sampling protocol was used (range of dissector number 20–70 per subfield). The volume of each subfield analysed per section was also calculated in each case. Analysis was carried out by one investigator (J.Z.) without knowledge of the pathological or clinical data relating to the patient.
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Quantitative analysis of dysgenetic features in medial temporal lobe
In this second part of the study, a total of 12 non-HS and 13 hippocampal sclerosis cases were included. The hippocampal formation was examined for any convolutional or ‘tectonic’ abnormality (Sloviter et al., 2004
). The presence of granule cell dispersion in the dentate gyrus was assessed in all cases. The pattern of any dispersion was categorized as diffuse, a bilayer pattern, or clusters of neurons in the molecular layer as described previously (Thom et al., 2002b). In addition, the breadth of the granule cell layer was estimated on each section by taking three measurements in the region of maximal dispersion using a Nikon DS 5 M camera attached to a Leica DMRB microscope and calculating the mean value in mm. On Nissl stained sections, using the adjacent LFB/Nissl stained section as a guide, the white matter of the parahippocampus gyrus (PHG) extending to the base of the sulcus between the fusiform gyrus and PHG and the border with the lateral ventricle was outlined in each case using the image analysis system, taking care not to include the cortex (Fig. 1B). In a pilot study of three cases, a 10% systematic random sample of this region, using the optical disector method, resulted in reproducible neuronal density measurements by a single observer (M.T.) (mean of number of dissectors between cases = 122; range = 37–328). All large neurons, as defined above, within this region were included. Similarly, the white matter in the core of the adjacent fusiform gyrus was also outlined away from the cortex and neurons counted within this whole area (mean of number of dissectors between cases = 176; range = 33–328). Neuronal densities per mm3 were calculated for each white matter area. For the PHG, the volume of the region was also calculated and the total number of neurons in the sample region estimated. Data analysis was carried out using SPSS version 11.5 for Windows (University College London) using the independent t-test or Mann–Whitney U-test to compare difference in neuronal densities or clinical factors between the groups. Correlations were carried out using Spearman's rank order or Pearson's correlation analysis. Differences in severity of granule cell dispersion between groups were assessed using ANOVA (analysis of variance).
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Hippocampal neuronal densities
In two cases, hippocampal sclerosis was bilateral (H2 and H8) and, in the remaining six cases, it was unilateral. In cases with unilateral hippocampal sclerosis, neuronal density measurements from the sclerotic hippocampus alone were used in the comparative analysis and, in cases with bilateral sclerosis, both sides were included (Table 4). The mean neuronal densities in hippocampal sclerosis cases were significantly lower compared with the control group and non-HS group for all subfields except for the subiculum (Table 4; P < 0.0001). The neuronal density reduction compared with controls was most pronounced for CA4 (84.3%) followed by CA1 (77.2%), CA3 (72.8%) and CA2 (52.8%). There was no significant difference in the mean neuronal density in any subfield for non-HS cases compared with controls. Volume measurements for each subfield also confirmed no significant reduction in any subfield in the non-HS group as compared with the controls (Table 4). The mean neuronal density for CA1 in the non-sclerotic side of unilateral HS cases was lower compared with the controls, but not significantly different (Table 4). Similarly, the differences in neuronal density for CA1 subfield between hemispheres were significantly greater in unilateral HS group than controls (P < 0.01), but not in the non-HS group compared with controls. There was no correlation between neuronal densities and post-mortem interval or fixation times for any cases or controls.
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IPIs, status epilepticus and age at first seizure
There was no difference in the presence of an IPI between the patient groups. Two patients in both the non-HS and HS groups had a history of an IPI on review of their notes; one patient with a prolonged convulsion and one with an episode of trauma was found in each group. Three patients in both the HS and non-HS group had episodes of status epilepticus recorded; the three patients in the non-HS group (N7, 8 and 9) did not have lower hippocampal neuronal densities compared with controls. The mean age of onset of epilepsy was 5.97 years in the non-HS group and 6.33 in the HS group; this was not significantly different (P = 0.95). Further analysis did not show any relationship between the age of onset of epilepsy and reduction of neuronal density in CA1 (P = 0.301), CA2 (P = 0.054), CA3 (P = 0.98) or CA4 (P = 0.398) in the HS group.
Duration of epilepsy, frequency and total number of seizures
All the patients in non-HS and HS groups had suffered from generalized tonic–clonic seizure (GTCS) for >20 years (Table 2), except for patients H6 and H8 who suffered only complex partial seizures. The majority of patients had focal epilepsy and any generalized seizures were secondary generalized seizures (based on EEG, clinical and imaging data) apart from two patients (N1, N3) who had probable idiopathic generalized epilepsy. The mean duration of epilepsy was 52.4 years (range 20–81 years) in the non-HS group and 61.7 years in the HS group ( range 37–84 years), which was not significantly different (P = 0.281). There was no significant correlation between duration of epilepsy and neuronal density in CA1 (P = 0.732), CA2 (P = 0.07), CA3 (P = 0.6) or CA4 (P = 0.84) in the HS group. The maximum frequency of GTCS was 44.6 per year in the non-HS group compared with 12.1 per year in the HS group; this was significantly different (P = 0.01). However, there was no correlation between frequency of GTCS and severity of neuronal loss in CA1 (P = 0.8), CA2 (P = 0.31), CA3 (P = 0.3) or CA4 (P = 0.86) in the HS group. Similarly, the minimum number of lifetime GTCSs was significantly higher in the non-HS group with mean total seizures of 1811 compared with 363 in the HS group (P = 0.007). For example, patient N9 had experienced >5000 GTCSs, but no hippocampal neuronal loss was seen. It was not possible in these patients to retrieve accurate data regarding the number of complex partial seizures.
Temporal lobe dysgenetic features
The 13 hippocampal sclerosis cases included in this part of the analysis included three patients with bilateral classical hippocampal sclerosis (H2, H11 and H10). In the remainder, unilateral classical hippocampal sclerosis was seen (H1,H3–6, H14 and H15), with two cases (H12 and H13), showing a mild end-folium gliosis on the less affected side. In unilateral hippocampal sclerosis cases, the sclerosed hippocampus was present on the left in four cases and on the right in six cases. Both left and right sides were included in the analysis in all hippocampal sclerosis cases. In the 12 cases included in the non-HS group, both left and right side were examined in five cases, the left only in four and the right side only in the remaining three cases due to a lack of available suitable tissue blocks representing all white matter regions.
The presence of granule cell dispersion was noted in 86% of the HS group. In eight cases, bilateral dispersion was noted—in five of these the granule cell layer being broader on the sclerotic side (Fig. 1H). In one case (H3), a bilaminar pattern was seen and, in another (H8), clusters of granule cells were present in the molecular layer. In a further case (H15), severe depletion of granule cells was observed. By contrast, very mild broadening of the granule cell layer or focal granule cell dispersion into the molecular layer was present in 43% of the non-HS cases and 30% of controls. The width of the granule cell layer showed a significant negative correlation with the density of neurons in CA1 (P = 0.032) and the mean width of the granule cell layer was highest in hippocampal sclerosis specimens (1.75 mm) compared with controls (0.96 mm). The mean values in hippocampi contralateral to hippocampal sclerosis was higher than in the non-HS group (1.4 mm versus 1.1 mm) (Table 5).
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There were significantly higher mean neuronal densities in all epilepsy cases compared with controls in the PHG white matter (1.59 versus 0.92x10–6/mm3; P < 0.003). There was no significant difference in mean neuronal densities in PHG white matter in HS cases compared with the side contralateral to hippocampal sclerosis or non-HS cases, but all were significantly different to controls (Table 5). When corrected for volumes, there were significantly fewer total PHG neuronal numbers in HS cases compared with non-HS cases. Comparing the left and right side in cases with unilateral hippocampal sclerosis, there was no significant difference in mean neuronal density, which was higher on the sclerotic side in only three out of 10 cases. In three cases (N9, N11 and N12), where well-classified cortical malformations were identified (polymicrogyria, tuberous sclerosis and focal cortical dysplasia Type IIa), there was no difference in PHG white matter neuronal density measurements compared with epilepsy cases without malformation. There was no correlation between fixation times and white matter neuronal densities.
Excessive convolutions in the CA1/subicular region were noted in six out of 13 of patients with hippocampal sclerosis (bilateral in three: H1, H5 and H10), in five out of 12 of the non-HS group (bilateral in three: N2, N3, N4 including one case reported previously by Thom et al., 2002a), and in two out of six controls (bilateral in one: C6) (Fig. 1 C–G). There were significantly higher mean white matter neuronal densities and total neuronal numbers in the PHG in epilepsy cases and controls with an abnormal hippocampal convolutional pattern (1.78 x 10–6/mm3 and 306, respectively) compared with those without (1.33 x 10–6/mm3 and 177, respectively; P < 0.05 and P < 0.01) (Fig. 2).
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White matter neuronal densities in the fusiform gyrus were higher than in the PHG for all patient groups and controls, but there was a significant correlation between PHG and fusiform gyrus neuronal densities for the epilepsy cases (P < 0.005). There was no significant difference between white matter neuronal densities in the fusiform gyrus of HS cases to non-HS cases or controls. Higher neuronal densities were not seen on the sclerotic side compared with preserved side in patients with unilateral hippocampal sclerosis.
In relation to clinical parameters, there was a positive correlation between the total number of neurons and neuronal density in the PHG white matter and the total number of seizures (P < 0.01), but not between the duration of seizures or the age of onset of epilepsy. There was no correlation between clinical features and granule cell dispersion.
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Discussion |
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How or when hippocampal sclerosis is acquired during the course of a patient's epilepsy history and the recognition of predisposing or risk factors are central areas in current epilepsy research. Identification of susceptible individuals could potentially lead to intervention and prevention of disease progression. Most human neuropathological studies over the last decades have concentrated on surgically removed tissues from patients with hippocampal sclerosis. In a recent International League Against Epilepsy (ILAE) report on hippocampal sclerosis, however, it was stated that the extent of the disease process could only properly be ascertained through autopsy studies, but that as autopsies were rare even in major epilepsy centres, this was unlikely to provide sufficient numbers of cases (Wieser, 2004
). In the current study, we have been able to carry out a quantitative neuropathological assessment of post-mortems from 28 patients from the Chalfont Centre for Epilepsy with long histories of well-characterized poorly controlled seizures. The main advantages with post-mortem tissue studies are the opportunities to: (i) address the effects of a lifetime of seizures on the hippocampus; (ii) compare findings between hemispheres; and (iii) investigate any underlying co-existing subtle developmental abnormalities within or beyond the temporal lobe.
There are likely to be several distinct causes of and groups of patients with hippocampal sclerosis (Cendes, 2004; Wieser, 2004). In many patients, hippocampal sclerosis is associated with the syndrome of mesial temporal lobe epilepsy (MTLE) where cellular loss combined with synaptic reorganization results in electrophysiological changes that lead to seizures; this group may represent ‘primary’ hippocampal sclerosis. In another group, hippocampal neuronal loss may be secondary to seizures themselves or due to an extra-hippocampal epileptogenic pathology. There is also a further clinical group of hippocampal sclerosis presenting with cognitive impairment alone in elderly patients without previous seizures (Leverenz et al., 2002); there is recent evidence suggesting this group may represent part of the spectrum of tauopathies or fronto-temporal dementias (Beach et al., 2003; Blass et al., 2004; Hatanpaa et al., 2004; Lippa and Dickson, 2004).
In the context of epilepsy, the exact temporal relationship between seizure history and hippocampal neuronal loss is not firmly established in the human condition. Findings from previous major studies conducted over the last 50 years correlating patterns of hippocampal damage with clinical parameters are summarized in Table 6 (including two previous post-mortem studies) and have shown contradictory findings. Our current post-mortem study has an added advantage that, as all patients were long-term residents at an epilepsy centre and under constant professional observation, more accurate records were available regarding the seizure history, in particular the precise number of lifetime generalized seizures.
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We selected cases from the pathology records on the basis of whether hippocampal sclerosis was present on qualitative inspection alone. Quantitative analysis, using design-based stereological probes, is regarded as essential to exclude any subtle neuronal loss in cases without apparent hippocampal sclerosis (West, 2002
). Using these methods in our study groups, we have confirmed the lack of any significant neuronal loss in our non-HS patient group compared with a control group without epilepsy. Our analysis also confirmed no difference in hippocampal volumes between these two groups; this potentially might have influenced neuronal density measurements. The density measurements were also not dependent on any variation in post-mortem or fixation times. Furthermore, from the clinical data, there was no evidence to support the view that the patients with hippocampal sclerosis had had more seizures than the non-HS group. In fact, the non-HS group had significantly higher mean seizure frequency and total number of lifetime generalized seizures. Our observations therefore suggest that, in a subgroup of patients with decades of poorly controlled epilepsy, hippocampal neurons are resistant to the effects of GTCS.
The pre-operative diagnosis of hippocampal sclerosis is based on the finding of hippocampal volume asymmetry on MRI as well as signal change. Evaluation and verification of neuronal loss in surgical material is naturally limited to the operated side. In a minority of patients who fail to have significant improvement from their seizures following hippocampectomy, there remains the possibility that subtle sclerosis of the remaining hippocampus exists. Post-mortem studies allow evaluation of both hippocampi and, in the present study, four of the 15 hippocampal sclerosis cases had qualitative evidence of bilateral hippocampal sclerosis. Similarly, previous post-mortem studies have confirmed the occurrence of bilateral hippocampal sclerosis (Babb, 1991) noted in up to 31% of a series of 55 epilepsy patients (Margerison and Corsellis, 1966). However, in the present study, stereological evaluation of the apparently non-sclerotic side in hippocampal sclerosis patients confirmed mean neuronal densities, including CA1 subfield, which were not significantly different from the control group, verifying contralateral hippocampal preservation.
Regarding the causes of hippocampal sclerosis, previous studies have suggested a high incidence of an initiating significant cerebral insult, more commonly referred to as IPIs (initial precipitating injuries), as a predisposing factor in patients with MTLE. The most common type of IPI recognized is a complex or prolonged febrile seizure reported in 30–60% of patients with hippocampal sclerosis (Lewis, 1999; Mathern et al., 2002; Baulac et al., 2004; Cendes, 2004). MRI studies following a febrile seizure have shown altered hippocampal signal consistent with oedema in the first 2 days (Scott et al., 2002) followed by hippocampal asymmetry appearing by 4–8 months (Scott et al., 2003) in support of their potential contribution to hippocampal sclerosis.
Data documenting IPIs may be subject to inaccuracies; incidents may not be always reported or recalled by the patients and conversely, in other cases, non-significant events may be ascribed IPI status (Wieser, 2004). We noted in the present study equal numbers of patients with histories of an IPI in the non-HS and hippocampal sclerosis group—although our numbers were small. Moreover, the timing of any initial insult as well as the onset of habitual seizures may be more relevant to any long-term effects on the hippocampus. In most clinical studies of patients with hippocampal sclerosis, the IPI typically occur early, before age 4 or 5 years (Mathern et al., 2002; Wieser, 2004). There is also experimental evidence that the very immature rodent hippocampus is resistant to early seizures (Riviello et al., 2002; Sutula et al., 2003), suggesting there is a narrow window of peak hippocampal vulnerability. The effects of seizures on the immature hippocampus are likely to be complex, influencing synaptogenesis, dentate neurogenesis and eventual susceptibility to epilepsy as well as neuronal loss (Holmes, 2002; Wasterlain et al., 2002). Indeed seizures themselves, under some conditions, may exert a protective effect on further neuron loss (Sutula et al., 2003). From previous studies, the age of habitual seizure onset is typically younger in epilepsy patients with hippocampal sclerosis than without (Table 6). In our present study with small numbers, we failed to identify any difference in the age of onset of seizures in post-mortem cases with and without hippocampal sclerosis.
Extensive loss of the hippocampal pyramidal neurons is well documented following an acute episode of status epilepticus (Sutula and Pitkanen, 2001) and may be unilateral (Men et al., 2000) or bilateral (Pohlmann-Eden et al., 2004). There is evidence from animal models that hippocampal neuronal loss may be ongoing following cessation of status (Pitkanen et al., 2002; Nairismagi et al., 2004). MRI volumetric studies of patients over 1 year following an episode of status, however, suggest that hippocampal volume loss is not invariable (Salmenpera et al., 2000). Similarly, in the present study we have confirmed cases with an absence of hippocampal neuronal loss at post-mortem examination, despite episodes of status epilepticus, including one patient with over 30 episodes recorded during her lifetime. The evidence in support of cumulative neuronal and hippocampal injury as a result of repeated brief seizures is less well-established (Sutula et al., 2003). In animal models, it has been shown that repeated brief seizures can lead to hippocampal neuronal loss in CA1 and CA4 (Kotloski et al., 2002). The experimental model type may influence whether recurrent seizures lead to neuronal loss (Holmes, 2002) as, in the post-status model, ongoing neuronal loss does not correlate with seizure number (Pitkanen et al., 2002). In humans, progressive hippocampal atrophy can be monitored in vivo by longitudinal MRI with volumetric analysis. There are case reports suggesting that hippocampal sclerosis can follow brief GTCS over a 3-year period (Briellmann et al., 2001). However, in other studies, including those of patients with newly diagnosed primary generalized and focal epilepsies, progressive MRI changes were not seen over a 3-year follow-up (Liu et al., 2002; Holtkamp et al., 2004). Retrospective, cross-sectional MRI studies have suggested increased hippocampal asymmetry or volume loss in patients with longer seizure duration in temporal lobe epilepsy (TLE) syndromes (Fuerst et al., 2001) or those with a greater total seizure number (Kalviainen and Salmenpera, 2002) but this did not apply to those with extra-temporal epilepsy (Salmenpera et al., 2001). Clinical support for progressive hippocampal pathology has been inferred from the ongoing deterioration that may occur in seizure control and cognitive function in some patients with hippocampal sclerosis (Fuerst et al., 2001; Wieser, 2004). The majority of previous pathological studies have also suggested a correlation between severity of hippocampal neuronal loss and the duration of epilepsy (Table 6). However, in only one previous study were the total number of seizures (both generalized and partial) estimated (Babb et al., 1984); this is likely to be a more accurate reflection of the patients' overall seizure burden and in this study, neither infact showed a correlation with hippocampal neuronal density. In the present study, we also found no relationship between the total number of generalized seizures (or their duration and frequency) and hippocampal neuronal loss. Although we did not have accurate clinical information of the number of complex partial seizures to correlate with pathology in these cases, our findings imply that a clear clinico-pathological relationship between progressive and cumulative neuronal depletion and ongoing generalized seizures is lacking.
There has been recent interest regarding the role or relative contribution of factors other than seizures (including genetic, environmental and developmental factors) in the development of hippocampal sclerosis. Hippocampal sclerosis is generally a sporadic condition, but there is evidence from some pedigrees that there may be common genetic basis for both febrile seizure and TLE, and that one or more genes may contribute to its development (Kobayashi et al., 2003; Baulac et al., 2004; Cendes, 2004). Polymorphisms in genes encoding inflammatory cytokines, implicated because of their potential role in modulating seizures and neuronal injury (Crespel et al., 2002; Vezzani et al., 2004), have been investigated including interleukin-1ß (Heils et al., 2000; Kanemoto et al., 2000, 2003; Buono et al., 2001) in addition to human leucocyte antigen (HLA) DR antigen type (Ozkara et al., 2002). The ApoE e4 genotype has an important influence on the vulnerability to many neurodegenerative and traumatic cerebral diseases, and an association with earlier onset TLE has been shown (Briellmann et al., 2000). In a recent study of >200 patients with TLE, however, we have not replicated any previously reported associations between gene polymorphisms and TLE phenotype (Cavalleri GL, Lynch JM, Depondt C, Burley M-W, Wood NW, Sisodiya SM, Goldstein DB, unpublished data). Regarding environmental factors contributing to hippocampal sclerosis, herpes virus infection (Donati et al., 2003) and head injury (Golarai et al., 2001; Maxwell et al., 2003) have been implicated. In the present study, early head injury was not identified as a risk factor and, although old cortical contusions were observed in a proportion of post-mortem cases (see Tables 1 and 3), these were considered to be more likely a sequel of epilepsy.
There has been growing speculation that underlying developmental abnormalities of the hippocampus predispose to or act as a template for hippocampal sclerosis (Blumcke et al., 2002; Wieser, 2004). In MRI studies, hippocampal abnormalities and asymmetries that may represent congenital abnormalities have been shown in children with febrile seizure (Fernandez et al., 1998; Grunewald et al., 2001; Scott et al., 2003), but the exact pathological correlate of these is unknown. Granule cell dispersion is a common finding in surgical series of hippocampal sclerosis, observed in 
40% of specimens (Thom et al., 2002b). Although initially considered to represent a developmental abnormality (Houser et al., 1990), it is now regarded more likely to be a consequence of the sclerosing process or the seizures themselves. Similar to observations in our previous study (Thom et al., 2002b), the severity of dispersion in these post-mortem cases showed a correlation with the severity of neuronal loss from CA1. Interestingly, in the present study, we also noted that granule cell dispersion was often seen bilaterally in the HS group, even where the hippocampal sclerosis was unilateral. The molecular signals influencing dispersion of granule cells away from the main layer may affect the rate of neurogenesis and radial gliosis in this region, possibly involving the reelin signalling pathway (Frotscher et al., 2003). Our observations therefore suggest that any altered cell signalling mechanism may involve both hippocampi, even though the neuronal loss and seizure focus is unilateral.
Structural abnormalities of hippocampal CA1 and subicular region in surgical specimens from patients with TLE have been demonstrated and termed ‘tectonic’ hippocampal formations due to the apparent distortion of the dentate gyrus by abnormal CA1 convolutions (Sloviter et al., 2004). Similar pathological lesions have been reported in patients with epilepsy having demonstrable hippocampal abnormalities on MRI (Baulac et al., 1998; Thom et al., 2002a). Excessive convolutions were noted in 44% of the epilepsy patients both with and without hippocampal sclerosis in the present study, but also in a third of the control group. The presence of milder tectonic abnormalities in non-epilepsy patients has been noted previously (Sloviter et al., 2004) and, although these lesions may be more prevalent in epilepsy, their exact significance in relation to seizures and indeed, hippocampal sclerosis is not yet uncertain.
In surgical resections of established hippocampal sclerosis, dissection of developmental from acquired microscopic changes is problematic, directing many studies to address the co-existence or not of subtle cortical malformations in the adjacent and better-preserved temporal neocortex. Identification of mild cortical dysplasias (previously termed microdysgenesis) (Palmini et al., 2004) in the lateral temporal lobe in patients with hippocampal sclerosis would be supportive of a common developmental aetiology. Quantitative studies on surgical tissues have demonstrated higher densities of white matter neurons, presumably representing heterotopic neurons, in the temporal lobe in hippocampal sclerosis cases compared with controls (Hardiman et al., 1988; Emery et al., 1997; Thom et al., 2001). In the present post-mortem study, we confirmed higher white matter neuronal densities in the epilepsy group compared with control groups. The density of white matter neurons in post-mortem hippocampal sclerosis cases (1.56x10–6/mm3 for the PHG) was comparable with our previous quantitative analysis of surgical hippocampal sclerosis specimens (1.10x10–6/mm3) (Thom et al., 2001). Differences in these observed values may be partly accounted for by the different anatomical sites studied (the superior to inferior temporal gyrus and deep white matter was included in surgical lobectomy cases), as well as the effects of different fixation and tissue processing methods on tissue shrinkage. The present post-mortem study has allowed comparison of identical anatomical regions (from both left and right hemipsheres) on tissues subjected to identical processing protocols and has demonstrated that greater white matter neuronal numbers are not confirmed on the side adjacent to hippocampal sclerosis, either when compared with the contralateral non-sclerotic side or to an epilepsy group without hippocampal sclerosis. In fact, in the fusiform gyrus, white matter neuronal densities were lower in the hippocampal sclerosis group compared with controls. This implies that an excess of white matter neurons is not invariably associated with hippocampal sclerosis. The overall higher white matter neuronal number observed in patients with epilepsy compared with controls may be a manifestation of seizures themselves due to excess neurogenesis or other trophic factors. Indeed, in the present study there was a trend for higher white matter neuron number with higher total lifetime seizure number.
In summary, we have confirmed that, in a proportion of patients with poorly controlled generalized seizures including episodes of status epilepticus throughout their lives, quantitative analysis of the hippocampal regions at post-mortem examination reveals an absence of neuronal loss. Furthermore, in patients with confirmed hippocampal sclerosis, no excess of microscopical cortical malformations was identified at post-mortem. This implies that recurrent seizures do not inevitably lead to hippocampal sclerosis and developmental abnormalities of the temporal lobe are not essential for its occurrence.
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Acknowledgements |
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We wish to thank consultant neurologists at the Chalfont Centre, M. Koepp, J.W.A.S. Sander and J. Duncan. The study was supported by a grant from the Wellcome Trust.
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References |
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