Neonatal death in mice lacking cardiotrophin-like cytokine is associated with multifocal neuronal hypoplasia.

Mice with null mutations of ciliary neurotrophic factor (Cntf) receptor alpha (Cntf-Ralpha), or cytokine-like factor 1 (Clf), one component of Cntf-II (a heterodimeric Cntf-Ralpha ligand), die as neonates from motor neuron loss affecting the facial nucleus and ventral horn of the lumbar spinal cord. Exposure to cardiotrophin-like cytokine (Clc), the other putative Cntf-II element, supports motor neuron survival in vitro and in ovo. Confirmation that Clc ablation induces an equivalent phenotype to Clf deletion would support a role for Clc in the functional Cntf-II complex. In this study, Clc knockout mice had decreased facial motility, did not suckle, died within 24 hours, and had 32% and 29% fewer motor neurons in the facial nucleus and lumbar ventral horn, respectively; thus, Clc is essential for motor neuron survival during development. The concordance of the Clc knockout phenotype with those of mice lacking Cntf-Ralpha or Clf bolsters the hypothesis that Clc participates in Cntf-II.

CD4+CD45RBHi T cell transfer induced colitis in mice is accompanied by osteopenia which is treatable with recombinant human osteoprotegerin.

BACKGROUND AND AIMS: Transfer of CD4+CD45RBHi T cells into semi syngeneic immunodeficient mice represents a model of inflammatory bowel disease (IBD). As patients with IBD often suffer from osteopenia, we studied if this T cell transfer in mice results in osteopenia in addition to colitis, and if treatment with osteoprotegerin (OPG) has effects on the bone mineral density of T cell transferred mice. We also investigated whether osteopenia was due to malabsorption as a result of a dysregulated digestive tract or as a consequence of the inflammatory process. METHODS: CD4+CD45RBHi or CD4+CD45RBLo T cells (4 x 10(5)) were sorted from CB6F1 and transferred into C.B.17 scid/scid mice. Recipient mice were treated with human IgG1 Fc (control) or Fc-OPG three times per week in a prophylactic regimen as well as a therapeutic regimen (after 10% body weight loss) and were evaluated for osteopenia and colitis. RESULTS: Mice that received CD4+CD45RBHi T cells developed osteopenia (as indicated by decreased bone density accompanied by decreased osteoblasts and increased osteoclasts) and colitis (as indicated by histological changes in the large intestine). Mice that received CD4+CD45RBLo T cells developed neither osteopenia nor colitis. All animals consumed, on average, the same amount of food and water over the course of the study. Prophylactic treatment with Fc-OPG increased bone density in mice that received either CD4+CD45RBHi or CD4+CD45RBLo T cells but had no effects on the gastrointestinal tract. Fc-OPG treatment of osteopenic mice with established IBD caused the normalisation of bone density. Osteopenia in CD4+CD45RBHi T cell recipients was accompanied by hypoparathyroidism that was partially normalised by treatment with Fc-OPG. CD4+CD45RBHi T cell recipients also had a bone marrow inflammatory cell infiltrate expressing tumour necrosis factor alpha which was unaffected by treatment with Fc-OPG. CONCLUSIONS: CD4+CD45RBHi T cell transfer results in osteopenia in addition to colitis. Evidence suggests that this osteopenia was induced by inflammatory cell infiltration and not by malabsorption of calcium. Recombinant human osteoprotegerin effectively treated the osteopenia. OPG may be a useful therapeutic option for treating osteopenia in patients with IBD.

Hippocampal N-methyl-D-aspartate receptor subunit mRNA levels in temporal lobe epilepsy patients.

Changes in the subunit stoichiometry of the N-methyl-D-aspartate (NMDA) receptor (NMDAR) alters its channel properties, and may enhance or reduce neuronal excitability in temporal lobe epilepsy patients. This study determined whether hippocampal NMDA receptor subunit mRNA levels were increased or decreased in temporal lobe epilepsy patients compared with nonseizure autopsy cases. Hippocampal sclerosis (HS; n = 16), non-HS (n = 10), and autopsy hippocampi (n = 9) were studied for NMDAR1 (NR1) and NR2A-D mRNA levels by using semiquantitative in situ hybridization techniques, along with neuron densities. Compared with autopsy hippocampi, non-HS and HS patients showed increased NR2A and NR2B hybridization densities per dentate granule cell. Furthermore, non-HS hippocampi showed increased NR1 and NR2B mRNA levels per CA2/3 pyramidal neuron compared with autopsy cases. HS patients, by contrast, showed decreased NR2A hybridization densities per CA2/3 pyramidal neuron compared with non-HS and autopsy cases. These findings indicate that chronic temporal lobe seizures are associated with differential changes in hippocampal NR1 and NR2A-D hybridization densities that vary by subfield and clinical-pathological category. In temporal lobe epilepsy patients, these findings support the hypothesis that in dentate granule cells NMDA receptors are increased, and excitatory postsynaptic potentials should be strongly NMDA mediated compared with nonseizure autopsies. HS patients, by comparison, showed decreased pyramidal neuron NR2A mRNA levels, and this suggests that NMDA-mediated pyramidal neuron responses should be reduced in HS patients compared with non-HS cases.

Hippocampal GABA and glutamate transporter immunoreactivity in patients with temporal lobe epilepsy.

OBJECTIVE: Sodium-coupled transporters remove extracellular neurotransmitters and alterations in their function could enhance or suppress synaptic transmission and seizures. This study determined hippocampal gamma-aminobutyric acid (GABA) and glutamate transporter immunoreactivity (IR) in temporal lobe epilepsy (TLE) patients. METHODS: Hippocampal sclerosis (HS) patients (n = 25) and non-HS cases (mass lesion and cryptogenic; n = 20) were compared with nonseizure autopsies (n = 8). Hippocampal sections were studied for neuron densities along with IR for glutamate decarboxylase (GAD; presynaptic GABA terminals), GABA transporter-1 (GAT-1; presynaptic GABA transporter), GAT-3 (astrocytic GABA transporter), excitatory amino acid transporter 3 (EAAT3; postsynaptic glutamate transporter), and EAAT2-1 (glial glutamate transporters). RESULTS: Compared with autopsies, non-HS cases with similar neuron counts showed: 1) increased GAD IR gray values (GV) in the fascia dentata outer molecular layer (OML), hilus, and stratum radiatum; 2) increased GAT-1 OML GVs; 3) increased astrocytic GAT-3 GVs in the hilus and Ammon's horn; and 4) no IR differences for EAAT3-1. HS patients with decreased neuron densities demonstrated: 1) increased OML and inner molecular layer GAD puncta; 2) decreased GAT-1 puncta relative to GAD in the stratum granulosum and pyramidale; 3) increased GAT-1 OML GVs; 4) decreased GAT-3 GVs; 5) increased EAAT3 IR on remaining granule cells and pyramids; 6) decreased glial EAAT2 GVs in the hilus and CA1 stratum radiatum associated with neuron loss; and 7) increased glial EAAT1 GVs in CA2/3 stratum radiatum. CONCLUSIONS: Hippocampal GABA and glutamate transporter IR differ in TLE patients compared with autopsies. These data support the hypothesis that excitatory and inhibitory neurotransmission and seizure susceptibility could be altered by neuronal and glial transporters in TLE patients.

Hippocampal AMPA and NMDA mRNA levels correlate with aberrant fascia dentata mossy fiber sprouting in the pilocarpine model of spontaneous limbic epilepsy.

There is considerable controversy whether aberrant fascia dentata (FD) mossy fiber sprouting is an epiphenomena related to neuronal loss or a pathologic abnormality responsible for spontaneous limbic seizures. If mossy fiber sprouting contributes to seizures, then reorganized axon circuits should alter postsynaptic glutamate receptor properties. In the pilocarpine-status rat model, this study determined if changes in alpha amino-3-hydroxy-5-methyl-4-isoxazole-propionate (AMPA) and n-methyl-D-aspartic acid (NMDA) receptor subunit mRNA levels correlated with mossy fiber sprouting. Sprague-Dawley rats were injected with pilocarpine (320 mg/kg; i.p.) and maintained in status epilepticus for 6 to 8 hours (pilocarpine-status). Rats were killed during the: (1) latent phase after neuronal loss but before spontaneous limbic seizures (day 11 poststatus; n = 7); (2) early seizure phase after their first seizures (day 25; n = 7); and (3) chronic seizure phase after many seizures (day 85; n = 9). Hippocampi were studied for neuron counts, inner molecular layer (IML) neo-Timm's staining, and GluR1-3 and NMDAR1-2b mRNA levels. Compared with controls, pilocarpine-status rats in the: (1) latent phase showed increased FD GluR3, NMDAR1, and NMDAR2b; greater CA4 and CA1 NMDAR1; and decreased subiculum GluR1 hybridization densities; (2) early seizure phase showed increased FD GluR3, increased CA1 NMDAR1, and decreased subiculum NMDAR2b densities; and (3) chronic seizure phase showed increased FD GluR2; increased FD and CA4 GluR3; decreased CA1 GluR2; and decreased subiculum GluR1, GluR2, NMDAR1, and NMDAR2b levels. In multivariate analyses, greater IML neo-Timm's staining: (1) positively correlated with FD GluR3 and NMDAR1 and (2) negatively correlated with CA1 and subiculum GluR1 and GluR2 mRNA levels. These results indicate that: (1) hippocampal AMPA and NMDA receptor subunit mRNA levels changed as rats progressed from the latent to chronic seizure phase and (2) certain subunit alterations correlated with mossy fiber sprouting. Our findings support the hypothesis that aberrant axon circuitry alters postsynaptic hippocampal glutamate receptor subunit stoichiometry; this may contribute to limbic epileptogenesis.

Altered hippocampal kainate-receptor mRNA levels in temporal lobe epilepsy patients.

This study determined whether hippocampal kainate (KA) receptor mRNA levels were increased or decreased in temporal lobe epilepsy patients compared with nonseizure autopsies. Hippocampal sclerosis (HS; n = 17), nonsclerosis (non-HS; n = 11), and autopsy hippocampi (n = 9) were studied for KA1-2 and GluR5-7 mRNA levels using semiquantitative in situ hybridization techniques, along with neuron densities. Compared with autopsy hippocampi, HS and non-HS cases showed decreased GluR5 and GluR6 hybridization densities per CA2 and/or CA3 pyramid. Furthermore, HS patients demonstrated increased KA2 and GluR5 hybridization densities per granule cell compared with autopsy hippocampi. These findings indicate that chronic temporal lobe seizures were associated with differential changes in hippocampal KA1-2 and GluR5-7 hybridization densities that vary by subfield and pathology group. In temporal lobe epilepsy patients, these results support the hypothesis that pyramidal cell GluR5 and GluR6 mRNA levels are decreased as a consequence of seizures, and in HS patients granule cell KA2 and GluR5 mRNA levels are increased in association with aberrant fascia dentata mossy fiber sprouting and/or hippocampal neuronal loss.

Hippocampal AMPA and NMDA mRNA levels and subunit immunoreactivity in human temporal lobe epilepsy patients and a rodent model of chronic mesial limbic epilepsy.

This study compared temporal lobe epilepsy patients, along with kindled animals and self sustained limbic status epilepticus (SSLSE) rats for parallels in hippocampal AMPA and NMDA receptor subunit expression. Hippocampal sclerosis patients (HS), non-HS cases, and autopsies were studied for: hippocampal AMPA GluR1-3 and NMDAR1&2b mRNA levels using in situ hybridization: GluR1, GluR2/3, NMDAR1, and NMDAR2(a&b) immunoreactivity (IR); and neuron densities. Similarly, spontaneously seizing rats after SSLSE, kindled rats, and control animals were studied for: fascia dentata neuron densities: GluR1 and NMDAR2(a&b) IR; and neo-Timm's staining. In HS and non-HS cases, the mRNA hybridization densities per granule cell, as well as molecular layer IR, showed increased GluR1 (relative to GluR2/3) and increased NMDAR2b (relative to NMDAR1) compared to autopsies. Likewise, the molecular layer of SSLSE rats with spontaneous seizures demonstrated more neo-Timm's staining, and higher levels of GluR1 and NMDAR2(a&b) IR compared to kindled animals and controls. These results indicate that hippocampal AMPA and NMDA receptor subunit mRNAs and their proteins are differentially increased in association with spontaneous, but not kindled, seizures. Furthermore, there appears to be parallels in fascia dentata AMPA and NMDA receptor subunit expression between HS (and non-HS) epileptic patients and SSLSE rats. This finding supports the hypothesis that spontaneous seizures in humans and SSLSE rats involve differential alterations in hippocampal ionotrophic glutamate receptor subunits. Moreover, non-HS hippocampi were more like HS cases than hippocampi from kindled animals with respect to glutamate receptors; therefore, hippocampi from kindled rats do not accurately model human non-HS cases, despite some similarities in neuron densities and mossy fiber axon sprouting.

Increased hippocampal AMPA and NMDA receptor subunit immunoreactivity in temporal lobe epilepsy patients.

This study determined if hippocampal AMPA and NMDA subunit immunoreactivity (IR) in temporal lobe epilepsy patients was increased compared with nonseizure autopsies. Hippocampi from hippocampal sclerosis patients (HS; n = 26) and nonsclerosis cases (non-HS: n = 12) were compared with autopsies (n = 6) and studied for GluR1, GluR2/3, NMDAR1, and NMDAR2 IR gray values (GV) along with fascia dentata and Ammon's horn neuron densities. Compared with autopsies, non-HS cases with similar neuron densities and HS patients with decreased neuron densities showed: (a) Increased GluR1 GVs in the fascia dentata molecular layer: (b) increased NMDAR1 GVs in the CA3-1 stratum radiatum and greater IR within pyramids; and (c) increased GluR2/3 and NMDAR2 GVs throughout all hippocampal subfields. Furthermore, HS patients showed that relative to the outer molecular layer: (a) GluR1 GV differences were decreased in the CA4/hilar region and CA1 stratum radiatum compared with autopsies; and (b) NMDAR2 GV differences were increased in the inner molecular layer compared with non-HS cases. In temporal lobe seizure patients, these results indicate that AMPA and NMDA receptor subunit IR was increased in HS and non-HS hippocampi compared with nonseizure autopsies. In humans, these findings support the hypothesis that glutamate receptor subunits are increased in association with chronic temporal lobe seizures, which may enhance excitatory neurotransmission and seizure susceptibility.

Anoxia during kainate status epilepticus shortens behavioral convulsions but generates hippocampal neuron loss and supragranular mossy fiber sprouting.

In rats, this study determined the impact of systemic hypoxia during late kainate-induced status epilepticus on hippocampal neuron loss and mossy fiber sprouting. Non-fasted Sprague Dawley rats were prepared as follows: Naive controls (n=5); rats placed 2 min in a hypoxia chamber (hypoxia only; n=6); rats that seized for more than 6 h from kainic acid (KA-status; 12 mg/kg; i.p.; n=7); and another KA-status group placed into the hypoxia chamber 75 min after the convulsions started (KA-status/hypoxia; n=16). All rats, except for half of the KA-status/hypoxia animals, were perfused 2 weeks later (short-term). The other 8 KA-status/hypoxia rats were perfused after 2 months (long-term). Hippocampal sections were studied for neuron densities and aberrant mossy fiber sprouting at three ventral to dorsal levels. Fascia dentata (FD) mossy fiber sprouting was quantified as an increase in the inner minus outer molecular layer (IML-OML) gray value (GV) difference. Behaviorally, KA-status/hypoxia rats had a shorter duration of convulsive status epilepticus than KA-status animals without anoxia. Hippocampal sections showed that compared to controls: (1) hypoxia-only rats showed no differences in ventral neuron densities and neo-Timm's stained IML-OML GVs; (2) KA-status rats had decreased CA3 densities and a non-significant increase in ventral IML-OML GV differences; and (3) KA-status/hypoxia short-term animals showed decreased hilar, CA3 and CA1 densities and increased ventral IML-OML GV differences. Compared to KA-status/hypoxia short-term rats, long-term animals showed no differences in ventral hippocampal neuron densities, but middle and dorsal sections demonstrated increased IML-OML GV differences and animals were observed to have spontaneous limbic epilepsy. These results indicate that rats exposed to kainate-induced status epilepticus for over 1 h and then a hypoxic insult had a shorter duration of convulsive status, decreased hippocampal neuron densities and greater FD mossy fiber sprouting than controls and the amount of neuronal damage and sprouting was slightly more than animals subjected to 6 h of kainate-induced status. This supports the hypothesis that a physiologic insult during status can shorten the convulsive episode, but still produce hippocampal pathology with a number of clinical and pathologic similarities to human mesial temporal lobe epilepsy (MTLE).

Human hippocampal AMPA and NMDA mRNA levels in temporal lobe epilepsy patients.

This study was designed to determine whether hippocampal neuronal AMPA (alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid) and NMDA (N-methyl-D-aspartate) mRNA levels were differentially increased in temporal lobe epilepsy patients compared with those measured in control tissue from non-seizure autopsies. Hippocampi from hippocampal sclerosis patients (n = 28) and temporal mass lesion cases (n = 12) were compared with those from the autopsies (n = 4), and studied for AMPA GluR1-3 and NMDAR1-2 mRNAs using semi-quantitative in situ hybridization, along with fascia dentata and Ammon's horn neuron densities. Compared with the autopsies, and without correction for neuron counts, the mass lesion cases with neuron densities similar to autopsies showed: (i) significantly increased NMDAR2 hybridization densities for fascia dentata granule cells; (ii) increased AMPA GluR3 mRNA densities for Ammon's horn pyramids; and (iii) similar or numerically increased mRNAs for all other subunits and hippocampal subfields. Compared with the autopsies, hippocampal sclerosis cases with decreased neuron densities showed: (i) significantly decreased AMPA GluR1-2 and NMDAR1-2 hybridization densities for Ammon's horn pyramids and (ii) similar or numerically decreased mRNAs for all other subunits and subfields. However, correcting for changes in neuron densities showed that hippocampal sclerosis patients had increased AMPA and NMDA mRNA levels per neuron compared with autopsies, and in the CA2 resistant sector GluR2 mRNA levels were numerically greater than autopsies and mass lesion cases. Furthermore, relative to autopsies both sclerosis and mass lesion hippocampi showed that, in the stratum granulosum, the greatest mRNA increases were in AMPA GluR1 and NMDAR2 compared with the other mRNAs. In chronic temporal lobe seizure patients these results indicate that mass lesion and sclerosis cases show differential increases in hippocampal AMPA and NMDA mRNA levels per neuron compared with autopsies, especially for AMPA GluR1 and NMDAR2 in fascia dentata granule cells. These findings support the hypothesis that temporal lobe seizures are associated with increased ionotropic glutamate receptor mRNA levels and alterations in receptor subunit composition that probably contribute to neuronal hyperexcitability, synchronization and seizure generation.

In contrast to kindled seizures, the frequency of spontaneous epilepsy in the limbic status model correlates with greater aberrant fascia dentata excitatory and inhibitory axon sprouting, and increased staining for N-methyl-D-aspartate, AMPA and GABA(A) receptors.

This study determined whether there were differences in hippocampal neuron loss and synaptic plasticity by comparing rats with spontaneous epilepsy after limbic status epilepticus and animals with a similar frequency of kindled seizures. At the University of Virginia, Sprague-Dawley rats were implanted with bilateral ventral hippocampal electrodes and treated as follows; no stimulation (electrode controls; n=5): hippocampal stimulation without status (stimulation controls; n=5); and limbic status from continuous hippocampal stimulation (n=12). The limbic status group were electrographically monitored for a minimum of four weeks. Four rats had no recorded chronic seizures (status controls), and all three control groups showed no differences in hippocampal pathology and were therefore incorporated into a single group (controls). Eight limbic status animals eventually developed chronic epilepsy (spontaneous seizures) and an additional eight rats were kindled to a similar number and frequency of stage 5 seizures (kindled) as the spontaneous seizures group. At the University of California (UCLA) the hippocampi were processed for: (i) Niss1 stain for densitometric neuron counts; (ii) neo-Timm's histochemistry for mossy fiber sprouting; and (iii) immunocytochemical staining for glutamate decarboxylase, N-methyl-D-aspartate receptor subunit 2, AMPA receptor subunit 1 and the GABA(A) receptor. In the fascia dentata inner and outer molecular layers the neo-Timm's stain and immunoreactivity was quantified as gray values using computer image analysis techniques. Statistically significant results (P<0.05) showed the following. Compared to controls and kindled animals, rats with spontaneous seizures had: (i) lower neuron counts for the fascia dentata hilus, CA3 and CA1 stratum pyramidale; (ii) greater supragranular inner molecular layer mossy fiber staining; and (iii) greater glutamate decarboxylase immunoreactivity in both molecular layers. Greater supragranular excitatory mossy fiber and GABAergic axon sprouting correlated with: (i) increases in N-methyl-D-aspartate receptor subunit 2 inner molecular layer staining; (ii) more AMPA receptor subunit 1 immunoreactivity in both molecular layers; and (iii) greater outer than inner molecular layer GABA(A) immunoreactivity. Furthermore, in contrast to kindled animals, rats with spontaneous seizures showed that increasing seizure frequency per week and the total number of natural seizures positively correlated with greater Timm's and GABAergic axon sprouting, and with increases in N-methyl-D-aspartate receptor subunit 2 and AMPA receptor subunit 1 receptor staining. In this rat limbic status model these findings indicate that chronic seizures are associated with hippocampal neuron loss, reactive axon sprouting and increases in excitatory receptor plasticity that differ from rats with an equal frequency of kindled seizures and controls. The hippocampal pathological findings in the limbic status model are similar to those in humans with hippocampal sclerosis and mesial temporal lobe epilepsy, and support the hypothesis that synaptic reorganization of both excitatory and inhibitory systems in the fascia dentata is an important pathophysiological mechanism that probably contributes to or generates chronic limbic seizures.

Human fascia dentata anatomy and hippocampal neuron densities differ depending on the epileptic syndrome and age at first seizure.

This study determined fascia dentata anatomy and hippocampal neuron densities in patients with different epileptic syndromes. Based on presurgical data, patients were classified into: (a) pediatric patients (n=19); (b) temporal mass lesion cases (n=14); and (c) hippocampal sclerosis patients (n=31). Surgically removed hippocampi and autopsies (n=34) were studied for: (a) hippocampal neuron densities; (b) stratum granulosum (SG) widths and lengths; and (c) hilar areas. The number of granule cells and hilar neurons per tissue section were estimated from the neuron densities and fascia dentata area measurements. Results showed that compared with autopsies (p<0.05): (a) pediatric patients had similar SG and hilar areas; granule cell density was lower (but not hilar neuron density); and the estimated number of granule cells was lower (but not the number of hilar neurons); (b) the widths of SG and hilar areas were greater in mass lesion cases; the density of granule cells and hilar neurons was lower; and the total estimated numbers of granule cells and hilar neurons were similar to those of the autopsies; and (c) hippocampal sclerosis patients had wider, yet shorter SG; hilar areas were smaller; granule cell and hilar densities were lower; and the total estimated numbers of granule cells and hilar neurons were lower than those of the autopsy cases. The duration of the seizures did not correlate with lower fascia dentata neuron densities or estimates of total granule cell and hilar neurons. Furthermore, greater SG widths correlated with lower hilar and CA4 neuron densities, but not with age at first seizure or duration of epilepsy. These results indicate that the size of the fascia dentata SG and hilus along with hippocampal neuron densities differ between surgical patients with different epileptic syndromes, and a wider SG was associated with a lower density of end folium neurons. These findings support the hypothesis that hippocampal sclerosis and granule cell dispersion are not the consequence of repetitive seizures beginning at an early developmental age, but seem to differ depending on the type of epileptic syndrome.

Granule cell mRNA levels for BDNF, NGF, and NT-3 correlate with neuron losses or supragranular mossy fiber sprouting in the chronically damaged and epileptic human hippocampus.

This study determined in temporal lobe epilepsy patients if there were correlations among hippocampal granule cell expression of neurotrophin mRNAs, aberrant supragranular mossy fiber sprouting, and neuron losses. Consecutive surgically resected hippocampi (n = 9) and comparison tissue from autopsies (n = 3) were studied for: 1. Granule cell mRNA levels using in situ hybridization for brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF), and neurotrophin-3 (NT-3); 2. neo-Timm supragranular mossy fiber sprouting; and 3. Ammon's horn neuron densities. Clinically, patients were classified into those with hippocampal sclerosis (HS; n = 7) and non-HS cases (i.e., mass lesions and autopsies; n = 5). Results showed that compared to non-HS cases, HS patients showed increased granule cell mRNA levels for BDNF, NGF, and NT-3 (p = 0.035, p = 0.04, p = 0.045 respectively; one-tail directional test). Moreover, granule cell BDNF mRNA levels correlated inversely with Ammon's horn neuron densities (p = 0.02) and correlated positively with greater supragranular mossy fiber sprouting (p = 0.02). NGF mRNA levels correlated inversely with Ammon's horn neuron densities (p = 0.02), and NT-3 mRNA levels correlated inversely with age at surgery (p = 0.04) and correlated positively with greater mossy fiber sprouting (p = 0.026). These results indicate in the chronically damaged human hippocampus that granule cells express neurotrophin mRNAs, and mRNA levels correlate with either hippocampal neuron losses or aberrant supragranular mossy fiber sprouting. These data support the hypothesis that in the epileptic human hippocampus, there may be pathophysiologic associations among mossy fiber synaptic plasticity, hippocampal neuron damage, and granule cell mRNA neurotrophin levels.

Glutamate AMPA receptors in the fascia dentata of human and kainate rat hippocampal epilepsy.

The present study examined the relationship between the patterns and densities of glutamate AMPA receptor sub-units GluR1 and GluR2/3 in the molecular layer of the fascia dentata and aberrant mossy fiber neoinnervation in human and kainate rat hippocampal epilepsy. Because AMPA sub-units modulate the fast glutamate synaptic transmission, we hypothesized that the AMPA receptor densities would be related to the glutamate-secreting mossy fibers, which could then contribute to seizure generation. In human hippocampal epilepsy, we found that the immunocytochemical labeling of GluR1 and GluR2/3 dendrites was positively related to the densities and spatial locations of the densest, aberrant neo-Timm stained supragranular mossy fibers. We used quantitative densitometry for the mossy fibers. However, the relatively faint and punctate immunocytochemical staining of the receptors did not allow true quantitative densitometry of the dendritic trees because in human epilepsy granule cell densities were decreased on average 50% of normal. Nevertheless, visual observations did confirm spatial relations between dense fascia dentata inner molecular layer mossy fibers and dense AMPA receptor staining. In the outer molecular layer, the mossy fibers were present only in the lower portion, were not densely-stained, and the AMPA receptors were only faintly-labeled. Nevertheless, outer molecular layer AMPA receptor densities were usually present more distally than were the mossy fibers. Experiments were done using intrahippocampal kainate epileptic rats to test the time courses for the changes in mossy fibers and AMPA receptors. The upregulation of inner and outer molecular layer AMPA receptors occurred maximally within 5 days post-kainate injection, prior to any mossy fiber supragranular ingrowth. One hundred and eighty days after ipsilateral kainate the AMPA receptors were increased bilaterally in the inner and outer molecular layers despite the fact that the contralateral aberrant supragranular mossy fibers were minor in comparison to the dense ipsilateral mossy fiber hyperinnervation. These results suggest that in hippocampal epilepsy AMPA receptor numbers increase throughout the length of the molecular layer dendrites; however the AMPA receptor densities are greater in rough relation to the greatest aberrant mossy fiber presynaptic inputs. Interestingly, the receptor upregulation precedes the mossy fiber ingrowth and may play a role in initiating axonal sprouting or in maintaining the aberrant mossy fiber synapses.

Neuron loss, mossy fiber sprouting, and interictal spikes after intrahippocampal kainate in developing rats.

This study determined neuron losses, mossy fiber sprouting, and interictal spike frequencies in adult rats following intrahippocampal kainic acid (KA) injections during postnatal (PN) development. KA (0.4 micrograms/0.2 microliters; n = 64) was injected into one hippocampus and saline into the contralateral side between PN 7 to 30 days. Animals were sacrificed 28 to 256 days later, along with age-matched naive animals (controls; n = 20). Hippocampi were studied for: (1) Fascia dentata granule cell, hilar, and CA3c neuron counts; (2) neo-Timm's stained supragranular mossy fiber sprouting; and (3) hippocampal and intracerebral interictal spike densities (n = 13). Mossy fiber sprouting was quantified as the gray value differences between the inner and outer molecular layer. Statistically significant results (p < 0.05) showed the following: (1) Compared to controls, CA3c and hilar neuron counts were reduced in KA-hippocampi with injections at PN 7-10 and PN 12-14 respectively and counts decreased with older PN injections. Granule cell densities on the KA-side and saline injected hippocampi were not reduced compared to controls. (2) In adult rats, supragranular mossy fiber sprouting was observed in 2 of 7 PN 7 injected animals. Compared to controls, increased gray value differences, indicating mossy fiber sprouting, were found on the KA-side beginning with injuries at PN 12-14 and increasing with older PN injections. On the saline-side only PN 30 animals showed minimal sprouting. (3) Mossy fiber sprouting progressively increased on the KA-side with longer survivals in rats injured after PN 15. Sprouting correlated positively with later PN injections and longer post-injection survival intervals, and not with reduced hilar or CA3c neuron counts. (4) On the KA-side, mossy fiber gray value differences correlated positively with in vivo intrahippocampal interictal spike densities. These results indicate that during postnatal rat development intrahippocampal kainate excitotoxicity can occur as early as PN 7 and increases with older ages at injection. This rat model reproduces many of the pathologic, behavioral, and electrophysiologic features of human mesial temporal lobe epilepsy, and supports the hypothesis that hippocampal sclerosis can be the consequence of focal injury during early postnatal development that progressively evolves into a pathologic and epileptic focus.

Childhood generalized and mesial temporal epilepsies demonstrate different amounts and patterns of hippocampal neuron loss and mossy fibre synaptic reorganization.

In this study, we determined whether childhood seizures were associated with hippocampal neuron loss and mossy fibre synaptic reorganization and if hippocampal sclerosis evolved from longer seizure histories. Children undergoing surgical treatment for catastrophic epilepsy were grouped into the following pathology categories: (i) those with generalized seizures and extra-hippocampal congenital pathologies (i.e. prenatal cortical dysplasia; n = 17); (ii) cases of generalized seizures and extra-hippocampal acquired lesions. (i.e. postnatal ischaemic injuries and encephalitis; n = 7); (iii) children with complex partial hippocampal epilepsy (n = 4). Further, to determine whether the epileptogenic location influenced hippocampal pathology, the seizure focus was classified as (i) hippocampal, (ii) temporal (n = 13) or (iii) extra-temporal (n = 11). Surgical and autopsy (n = 23) hippocampi were studied for (i) fascia-dentata (FD) and Ammon's horn (AH) neuron densities; (ii) thickness; height or length of the FD molecular layer, stratum granulosum (SG) and stratum pyramidale; and (iii) grey value (GV) densities of supragranular neo-Timm's staining. Statistically significant results (P < 0.05) showed the following. (i) Autopsy hippocampal neuron densities for the hilus (H), AH and prosubiculum (Pro) decreased logarithmically at the same time as the thickness of the stratum pyramidale and Pro increased. By contrast, autopsy granule cell densities and thickness did not significantly change with age; however, the SG lengthened-expanding around the enlarging H. Further, the supragranular molecular layer height increased logarithmically, and took longer than the increase in stratum pyramidale thickness. (ii) Compared with age-matched autopsies, young children with a history of hippocampal seizures showed decreased granule cell, hilar and regio superior neuron densities similar to adults with hippocampal sclerosis (average loss 70%). By contrast, children with extra-hippocampal congenital or acquired pathologies showed only decreased granule cell densities, along with a thinner and shorter SG. Compared with extra-temporal locations, those with temporal lobe lesions showed decreased hilar and AH neuron densities, but averaged 20-30% less than autopsies and not in the pattern typical of hippocampal sclerosis. (iii) The neo-Timm's GV densities, when compared with autopsies, showed supragranular mossy fibre sprouting in children with congenital pathologies and temporal lobe lesions; however, the greatest GVs were in children with hippocampal seizures. (iv) Of the children with extra-hippocampal congenital or acquired pathologies there were no statistical correlations between longer duration of seizures with changes in neuron densities, hippocampal heights, or mossy fibre sprouting. These results indicate the following. (i) In the human there is anatomical evidence for postnatal maturation of the hippocampus and our results are consistent with the notion that AH pyramids are a stable population; however, there are probably increases in granule cell numbers. Further, compared with the AH, dendritic maturation of the FD granule cells appears to take longer. (ii) Extra-hippocampal childhood seizures whether from prenatal or postnatal aetiologies are associated with moderate FD and minimal AH neuron losses and signs of aberrant mossy fibre sprouting. (iii) By contrast, young children with the syndrome of mesial temporal epilepsy show patterns of neuron loss and mossy fibre sprouting, typical of hippocampal sclerosis. (iv) Repeated extra-hippocampal childhood seizures are not associated with progressive evolution of hippocampal damage or mossy fibre sprouting. These findings support the hypothesis that childhood seizures can damage or alter the postnatally developing granule cells of the human hippocampus, and that early neuron loss and aberrant axon circuits may contribute to chronic hippocampal seizures. However, repeated childhood generalized seiz

Aberrant hippocampal mossy fiber sprouting correlates with greater NMDAR2 receptor staining.

This study determined in temporal lobe epilepsy patients and rats injected with intrahippocampal kainate (KA) whether fascia dentata molecular layer mossy fiber sprouting was associated with increases in NMDAR2 immunoreactivity (IR). Patients with hippocampal sclerosis (n = 11) were compared with those with temporal mass lesions (n = 7) and material obtained at autopsies (n = 4); and unilateral KA-injected rat hippocampi (n = 7) were compared with the contralateral saline-injected side and non-lesioned animals (n = 7; control). Hippocampi were studied for neo-Timm's stained mossy fiber sprouting and NMDAR2 IR. The staining was quantified as gray values (GV) using computer image analysis. Hippocampal sclerosis patients and KA-injected rats showed the greatest inner molecular layer (IML) mossy fiber sprouting and NMDAR2 staining. Compared with autopsies and patients with mass lesions, hippocampal sclerosis patients had greater IML neo-Timm's (p = 0.0018) and NMDAR2 staining (p = 0.0063). Similarly, compared with controls and saline-injected rats, KA-injected hippocampi showed greater IML mossy fiber sprouting and NMDAR2 IR (p = 0.0001). Furthermore, IML mossy fiber sprouting positively correlated with greater IML NMDAR2 staining in both human and experimental rat groups (p < 0.0099). These results support the hypothesis that in severely damaged hippocampi abnormal mossy fiber sprouting and concordant increases in IML NMDAR2 receptor staining may contribute or partially explain granule cell hyperexcitability and the pathophysiology of hippocampal epilepsy.

The pathophysiologic relationships between lesion pathology, intracranial ictal EEG onsets, and hippocampal neuron losses in temporal lobe epilepsy.

In temporal lobe epilepsy (TLE) lesion patients the pathology, location of intracranial ictal EEG onsets, and hippocampal neuron losses were compared. Patients (n = 63) were classified into: (1) Tumors (n = 26, e.g. astrocytomas, gangliogliomas); (2) vascular (n = 9, e.g. cavernous and venous angiomas); (3) developmental (n = 17, e.g. cortical dysplasia, heterotopias); or (4) atrophic (n = 11, e.g. cortical or white matter encephalomalacia). Other variables were; (1) the location of the temporal lesion in the mesial to lateral, and anterior to posterior plane, (2) a clinical history of an initial precipitating injury (IPI) prior to the onset of TLE (e.g. prolonged first seizure, head trauma), (3) hippocampal neuron densities, (4) focal or regional location by intracranial depth EEG of ictal onsets, and (5) seizure outcomes. Results showed that severe hippocampal neuron losses were associated with two statistically significant findings. First, patients with mesial lesions in or adjacent to the body of the hippocampus had greater neuron losses compared to mesial lesions anterior or posterior to the hippocampus (P = 0.04). Second, lesion patients with an IPI history had greater Ammon's horn (AH) neuron losses compared to those without IPI histories (P = 0.0005), and the profile of loss was similar to hippocampal sclerosis (HS). Granule cell losses correlated in a complex manner in that; 1) by regression analysis densities decreased with longer intervals of TLE (P = 0.006), (2) tumor patients with IPIs had less granule cell loss compared to those without IPIs intervals of TLE (P = 0.006), (2) tumor patients with IPIs had less granule cell loss compared to those without IPIs (P = 0.05), and (3) developmental patients with IPIs had greater granule cell loss than patients without IPIs (P = 0.009). Mesial-temporal depth EEG electrodes were the first areas of ictal activity in 15 of 16 patients (94%), and greater hippocampal neuron losses were not associated with focal mesial-temporal EEG onsets. Seizure outcomes were worse in tumor patients compared to HS patients (P = 0.01), and patients with post-resection seizures had incomplete resections of their lesions and/or hippocampi. These results indicate that in TLE lesion patients the amount and pattern of hippocampal neuron loss depends on the location of the lesion, the pathologic classification, and a history of an IPI. Further, despite variable neuron losses, in temporal lesion patients the hippocampus was nearly always involved in the genesis or propagation of the chronic seizures.