Kerry W. Thompson

Genetically engineered GABA-producing cells demonstrate anticonvulsant effects and long-term transgene expression when transplanted into the central piriform cortex of rats.

Local application of GABA-potentiating agents can prevent or reduce the development and maintenance of behavioral seizures induced by limbic kindling in rats. Microinjection and lesion studies suggest that the transition zone between anterior and posterior piriform cortex (PC), termed here central PC, is a potential target for transplantation of GABA-producing cells. In the present study, we transplanted conditionally immortalized mouse cortical neurons, engineered with the GABA-synthesizing enzyme GAD(65), to the central PC of rats. Suspensions of 1.5 x 10(5) cells in 1 microl were transplanted bilaterally. Control animals received transplantation of beta-galactosidase (beta-gal)-expressing cells. All rats were subsequently kindled through a chronically implanted electrode placed in the basolateral amygdala. The pre- and postkindling threshold currents for eliciting behavioral seizures were determined before and after kindling. We found the prekindling partial seizure threshold to be significantly increased by about 200% in the rats that received the GABA-producing cells compared to rats receiving beta-gal-producing transplants. After kindling, the seizure threshold tended to be higher by 100% in rats that received GABA-producing cells, although the difference from controls was not statistically significant. GABA-producing transplants had no significant effect on the rate of amygdala kindling, but the latency to the first generalized seizure during kindling was significantly increased in animals receiving GABA-producing cells. The transplanted cells showed long-term GAD(65) expression as verified immunohistologically after termination of the experiments. The findings substantiate and extend previous findings that the central PC is part of the anatomical substrate that facilitates propagation from partial to generalized seizures. The data demonstrate that genetically engineered cells have the potential to raise seizure thresholds when transplanted to the central PC.

Conditionally immortalized cell lines, engineered to produce and release GABA, modulate the development of behavioral seizures.

Transplantation of genetically engineered cells can provide sustained focal delivery of naturally occurring molecules, including neurotransmitters and growth factors. We have engineered immortalized mouse cortical neurons and glia to deliver GABA by driving GAD(65) expression. Engineered cell lines showed GAD(65) mRNA expression, enzymatic activity, and GABA release. In vitro, basal flux of GABA was approximately 20% of total cellular GABA. We transplanted these GABA-producing cells bilaterally into either the anterior or the posterior substantia nigra of 43 rats. The rats were subsequently kindled through an electrode placed in the entorhinal cortex. GABA-producing cells, but not beta-galactosidase-producing cells, affected kindling rates. The number of stimulations needed to reach the first stage-5 seizure and to achieve full kindling differed significantly between the anterior and posterior transplantation sites when GAD(65)-producing cells were transplanted but not when beta-galactosidase-producing cells were transplanted. Our data show that transplanted engineered cells can make and release GABA at physiologically meaningful concentrations.

Treatment of Experimental Status Epilepticus in Immature Rats: Dissociation Between Anticonvulsant and Antiepileptogenic Effects

We studied the effects of treating status epilepticus (SE) induced by lithium and pilocarpine at postnatal day 15 (P15) or 28 (P28), on the severity of acute SE and of SE-induced epileptogenesis. Rats received topiramate (10 or 50 mg/kg, IP) or diazepam (5 mg/kg, IP) 20, 40 or 70 min after pilocarpine, and three months after SE 24-h video/EEG recordings were obtained for one (P28) or two weeks (P15) continuously.In P15 rats, topiramate did not modify the course of SE, yet treatment at 20 or 40 min completely prevented the development of spontaneous recurrent seizures (SRS) while later treatment (70 min) was partially effective in reducing the severity and frequency of SRS. Diazepam was effective against acute SE at all time points tested. Early (20 min) but not late treatment with diazepam had the effect of reducing the frequency and severity of SRS.In P28 rats, both drugs reduced the cumulative seizure time. Early treatment (20 min) with either drug reduced the incidence of chronic epilepsy. Late treatment (40/70 min) did not alter the incidence of SRS, but decreased their frequency. This study demonstrates that, in the treatment of SE, anticonvulsant and antiepileptogenic effects can be dissociated in a development-specific manner: topiramate was antiepileptogenic without being an effective anticonvulsant in P15 animals at the doses tested. Diazepam, on the other hand, was a better anticonvulsant than an antiepileptogenic agent in the P15 animals at the dose tested. Such effects were not seen in the older animals.

Genetically engineered cells with regulatable GABA production can affect afterdischarges and behavioral seizures after transplantation into the dentate gyrus.

Intractable seizures originating in the mesial temporal lobe can often be controlled by resection. An alternative to removing hippocampal tissue may be transplantation of GABA-producing cells. Neural cell transplantation has been performed in hundreds of patients, including some with temporal lobe epilepsy. This study evaluates the seizure-suppressing capabilities of engineered GABA-producing cells transplanted into the dentate gyrus. Immortalized neurons were engineered to produce GABA under the control of doxycycline. The cells were characterized for GABA production in vitro and for their ability to raise GABA concentrations in vivo. Cells were transplanted bilaterally into the dentate gyrus of rats and tested in two separate paradigms. Afterdischarge thresholds and durations were tested with granule cell stimulation, and the development of behavioral seizures, induced by daily electrical stimulation of the major excitatory input pathway into the dentate gyrus, was assessed in the presence, or the absence, of doxycycline. GABA production was under the tight control of doxycycline. Cells engineered to produce GABA raised tissue GABA concentrations in the hippocampus compared with non GABA-producing cells, and this was abolished when doxycycline was administered. GABA-producing cells raised the threshold, and shortened the duration of hippocampal afterdischarges elicited by granule cell stimulation. Lastly, the appearance of stage 5 seizures was slowed in the kindling paradigm, compared with a group that received non-GABA-producing cells, and compared with a group that received GABA-producing cells but was administered doxycycline. This study shows that targeted hippocampal implants of genetically engineered cells have the potential to raise GABA levels and to affect seizure development. The ability to suppress the production of GABA, and to modulate the physiological effects of the transplanted cells provides an important level of experimental control. These techniques, combined with stem cell technology, may advance cell-based therapies for epilepsy and other diseases of the CNS.

Transplants of Cells Engineered to Produce GABA Suppress Spontaneous Seizures

Summary:  Purpose: Cell transplantation into the brain is an aggressive clinical alternative. The hopes of treating diseases like intractable temporal lobe epilepsy have been subdued because the preclinical successes thus far have shown only slowing of epileptogenesis, or suppression of electrically induced seizures. Because the hallmark of epilepsy is spontaneous seizures, the clinical relevance of these studies has been questioned. The purpose of this study was to establish that cells genetically engineered to produce γ‐aminobutyric acid (GABA) could suppress spontaneous seizures in an accepted model of temporal lobe epilepsy.

Seizure-induced Neuronal Death in the Immature Brain

The response of the developing brain to epileptic seizures and to status epilepticus is highly age-specific. Neonates with their low cerebral metabolic rate and fragmentary neuronal networks can tolerate relatively prolonged seizures without suffering massive cell death, but severe seizures in experimental animals inhibit brain growth, modify neuronal circuits, and can lead to behavioral deficits and to increases in neuronal excitability. Past infancy, the developing brain is characterized by high metabolic rate, exuberant neuronal and synaptic networks and overexpression of receptors and enzymes involved in excitotxic mechanisms. The outcome of seizures is highly model-dependent. Status epilepticus may produce massive neuronal death, behavioral deficits, synaptic reorganization and chronic epilepsy in some models, little damage in others. Long-term consequences are also highly age- and model-dependent. However, we now have some models which reliably lead to spontaneous seizures and chronic epilepsy in the vast majority of animals, demonstrating that seizure-induced epileptogenesis can occur in the developing brain. The mode cell death from status epilepticus is largely (but not exclusively) necrotic in adults, while the incidence of apoptosis increases at younger ages. Seizure-induced necrosis has many of the biochemical features of apoptosis, with early cytochrome release from mitochondria and capase activation. We speculate that this form of necrosis is associated with seizure-induced energy failure.

Hippocampal Stimulation Produces Neuronal Death in the Immature Brain

We re-examined the proposed resistance of the immature brain to seizure-induced damage. In awake, freely moving rat pups, intermittent perforant path stimulation produced selective hippocampal cell loss and reduction in paired-pulse inhibition. During 16 h of stimulation, animals showed frequent wet dog shakes and hind-limb scratching movements but no convulsive motor activity. In situ end-labelling performed 2 h after the end of stimulation showed an intense band of positively-labelled eosinophilic cells with condensed profiles bilaterally in the dentate granule cell layer of stimulated animals. Control animals showed no in situ end-labelling positivity in the dentate gyrus. These cells were not observed 24 h later, suggestive of rapidly scavenged apoptotic cells. One day after the end of stimulation, many necrotic interneurons with eosinophilic cytoplasm and pyknotic nuclei were observed in the hilus of the stimulated dentate gyrus in all rats tested. Hippocampal pyramidal cells in CA1, CA3 and subiculum showed bilateral damage greater on the side of stimulation, and prepiriform cortex sustained bilateral symmetrical lesions. One month after perforant path stimulation, Cresyl Violet staining showed the number of large hilar interneurons (>15 microm) was reduced on the stimulated side (54.1 +/- 12.2) compared to the non-stimulated side (100.5 +/- 10.2 cells, P<0.01). Immunohistochemical analysis showed significant losses in somatostatin (8.5 +/- 1.6 stimulated side, 22.8 +/- 3.8 unstimulated side, P<0.05) and neuropeptide Y (12.8 +/- 3.2 stimulated side, 17.0 +/- 4.1 unstimulated side, P<0.05) immunoreactive cells in the stimulated hilus but no loss of parvalbumin-immunoreactive cells. Significant reductions in paired-pulse inhibition were found after stimulation but there was some return of inhibition by one month. These combined data demonstrate that the immature brain can incur damage as a result of prolonged seizure-like hippocampal activity mimicking status epilepticus in immature rats. The hippocampal damage produced by perforant path stimulation is associated with the immediate loss of physiological inhibition suggesting important modification of excitatory control in an extremely epileptogenic region of the brain.

Self-sustaining Status Epilepticus: A Condition Maintained by Potentiation of Glutamate Receptors and by Plastic Changes in Substance P and Other Peptide Neuromodulators

Summary: We describe a model of self‐sustaining status epilepticus (SSSE) induced by stimulation of the perforant path in free‐running rats. In this model, seizures can be transiently suppressed by intrahippocampal injection of a blocker of α‐amino‐3‐hydroxy‐5‐methylisoxazole‐4‐propionic acid/kainate synapses but return in the absence of further stimulation when the drug ceases to act. However, seizures are irreversibly abolished by blockers of N‐methyl‐d‐aspartate receptors given locally or systemically. SSSE is enhanced by substance P and its agonists and blocked by its antagonists. SSSE induces novel expression of substance P‐like immunoreactivity in hippocampal principal cells. These changes and those in other limbic peptides may contribute to the maintenance of SSSE and to the modulation of hippocampal excitability during epileptic seizures.

Rational polytherapy in the treatment of acute seizures and status epilepticus.

We used a model of severe cholinergic status epilepticus (SE) to study polytherapy aimed at reversing the effects of seizure‐induced loss of synaptic GABAA receptors and seizure‐induced gain of synaptic NMDA receptors. Combinations of a benzodiazepine with ketamine and valproate, or with ketamine and brivaracetam, were more effective and less toxic than benzodiazepine monotherapy in this model of SE.

Transplantation of GABA-producing cells for seizure control in models of temporal lobe epilepsy

SummaryA high percentage of patients with temporal lobe epilepsy (TLE) are refractory to conventional pharmacotherapy. The progressive neurodegenerative processes associated with a lifetime of uncontrolled seizures mandate the development of alternative approaches to treat this disease. Transplantation of inhibitory cells has been suggested as a potential therapeutic strategy to achieve seizure suppression in humans with intractable TLE. Preclinical investigations over 20 years have demonstrated that multiple cell types from several sources can produce anticonvulsant, and antiepileptogenic, effects in animal models of TLE. Transplanting GABA-producing cells, in particular, has been shown to reduce seizures in several well-established models. This review addresses experimentation using different sources of transplantable GABAergic cells. highlighting progress with fetal tissue, neural cell lines, and stem cells. Regardless of the source of the GABAergic cells used in seizure studies, common challenges have emerged. Several variables influence the anticonvulsant potential of GABA-producing cells. For example, tissue availability, graft survival, immunogenicity, tumorigenicity, and varying levels of cell migration, differentiation, and integration into functional circuits and the microenvironment provided by sclerotic tissue all contribute to the efficacy of transplanted cells. The challenge of understanding how all of these variables work in concert, in a disease process that has no well-established etiology, suggests that there is still much basic research to be done before rational cell-based therapies can be developed for TLE.