Promoting bioengineered tooth innervation using nanostructured and hybrid scaffolds.

The innervation of teeth mediated by axons originating from the trigeminal ganglia is essential for their function and protection. Immunosuppressive therapy using Cyclosporine A (CsA) was found to accelerate the innervation of transplanted tissues and particularly that of bioengineered teeth. To avoid the CsA side effects, we report in this study the preparation of CsA loaded poly(lactic-co-glycolic acid) (PLGA) nanoparticles, their embedding on polycaprolactone (PCL)-based scaffolds and their possible use as templates for the innervation of bioengineered teeth. This PCL scaffold, approved by the FDA and capable of mimicking the extracellular matrix, was obtained by electrospinning and decorated with CsA-loaded PLGA nanoparticles to allow a local sustained action of this immunosuppressive drug. Dental re-associations were co-implanted with a trigeminal ganglion on functionalized scaffolds containing PLGA and PLGA/cyclosporine in adult ICR mice during 2weeks. Histological analyses showed that the designed scaffolds did not alter the teeth development after in vivo implantation. The study of the innervation of the dental re-associations by indirect immunofluorescence and transmission electron microscopy (TEM), showed that 88.4% of the regenerated teeth were innervated when using the CsA-loaded PLGA scaffold. The development of active implants thus allows their potential use in the context of dental engineering. STATEMENT OF SIGNIFICANCE: Tooth innervation is essential for their function and protection and this can be promoted in vivo using polymeric scaffolds functionalized with immunosuppressive drug-loaded nanoparticles. Immunosuppressive therapy using biodegradable nanoparticles loaded with Cyclosporine A was found to accelerate the innervation of bioengineered teeth after two weeks of implantation.

Intact neurobehavioral development and dramatic impairments of procedural-like memory following neonatal ventral hippocampal lesion in rats.

Neonatal ventral hippocampal lesions (NVHL) in rats are considered a potent developmental model of schizophrenia. After NVHL, rats appear normal during their preadolescent time, whereas in early adulthood, they develop behavioral deficits paralleling symptomatic aspects of schizophrenia, including hyperactivity, hypersensitivity to amphetamine (AMPH), prepulse and latent inhibition deficits, reduced social interactions, and spatial working and reference memory alterations. Surprisingly, the question of the consequences of NVHL on postnatal neurobehavioral development has not been addressed. This is of particular importance, as a defective neurobehavioral development could contribute to impairments seen in adult rats. Therefore, at several time points of the early postsurgical life of NVHL rats, we assessed behaviors accounting for neurobehavioral development, including negative geotaxis and grip strength (PD11), locomotor coordination (PD21), and open-field (PD25). At adulthood, the rats were tested for anxiety levels, locomotor activity, as well as spatial reference memory performance. Using a novel task, we also investigated the consequences of the lesions on procedural-like memory, which had never been tested following NVHL. Our results point to preserved neurobehavioral development. They also confirm the already documented locomotor hyperactivity, spatial reference memory impairment, and hyperresponsiveness to AMPH. Finally, our rseults show for the first time that NVHL disabled the development of behavioral routines, suggesting dramatic procedural memory deficits. The presence of procedural memory deficits in adult rats subjected to NHVL suggests that the lesions lead to a wider range of cognitive deficits than previously shown. Interestingly, procedural or implicit memory impairments have also been reported in schizophrenic patients.

Modifications of local cerebral glucose utilization in thalamic structures following injection of a dopaminergic agonist in the nucleus accumbens--involvement in antiepileptic effects?

Dopaminergic transmission in the nucleus accumbens (NAcc) is implicated in different aspects of reward and motivational mechanisms. More recently, it has been suggested that this nucleus could also be involved in the modulation of generalized epileptic seizures. In particular, microinjection of dopaminergic agonists in the NAcc suppresses the occurrence of epileptic seizures in a model of absence seizures, the GAERS (generalized absence epileptic rats from Strasbourg). The aim of this study was to identify the structures involved in this effect. Local cerebral metabolic rates for glucose utilization (LCMRglc) were measured in different parts of the basal ganglia and output structures after apomorphine injection in the NAcc in GAERS and in the inbred non-epileptic rats (NE), concomitantly with seizure suppression. Apomorphine injection in the NAcc induced a significant increase of glucose intake in the anteromedial, mediodorsal and ventrolateral nuclei of the thalamus in NE rats, while no significant changes were observed in the basal ganglia structures (globus pallidus, subthalamic nucleus, substantia nigra). Furthermore, microinjections of muscimol (100 and 200 pmol/side) in the mediodorsal nucleus of the thalamus in GAERS rats suppressed seizures. These results suggest that the mediodorsal nucleus of the thalamus could be involved in absence seizures modulation. Along with data from the literature, our data suggest that this nucleus could participate in the control of the basal ganglia over generalized epileptic seizures.

Neuroprotective properties of topiramate in the lithium-pilocarpine model of epilepsy.

The lithium-pilocarpine model reproduces the main characteristics of human temporal lobe epilepsy. After status epilepticus (SE), rats exhibit a latent seizure-free phase characterized by development of extensive damage in limbic areas and occurrence of spontaneous recurrent seizures. Neuroprotective and antiepileptogenic effects of topiramate were investigated in this model. SE was induced in adult male rats by LiCl (3 mEq/kg) followed 20 h later by pilocarpine (25 mg/kg). Topiramate (10, 30, or 60 mg/kg) was injected at 1 and 10 h of SE. Injections were repeated twice a day for six additional days. Another group received two injections of diazepam on the day of SE and of vehicle for 6 days. Neuronal damage was assessed at 14 days after SE by cell counting on thionin-stained sections. Occurrence of spontaneous recurrent seizures (SRS) was videorecorded for 10 h per day in other groups of rats. In diazepam-treated rats, the number of neurons was dramatically reduced after SE in all subregions of hippocampus and layers II-IV of ventral cortices. At all doses, topiramate induced a 24 to 30% neuroprotection in layer CA1 of hippocampus (p < 0.05). In CA3b, the 30-mg/kg dose prevented neuronal death. All rats subjected to SE became epileptic. The latency (14-17 days) to and frequency of SRS were similar in topiramate- and diazepam-treated rats. The high mortality in the 30 mg/kg topiramate group (84%) was possibly the result of interaction between lithium and topiramate. In conclusion, topiramate displayed neuroprotective properties only in CA1 and CA3 that were not sufficient to prevent epileptogenesis.

Vigabatrin protects against hippocampal damage but is not antiepileptogenic in the lithium-pilocarpine model of temporal lobe epilepsy.

In temporal lobe epilepsy (TLE), the nature of the structures involved in the development of the epileptogenic circuit is still not clearly identified. In the lithium-pilocarpine model, neuronal damage occurs both in the structures belonging to the circuit of initiation and maintenance of the seizures (forebrain limbic system) as well as in the propagation areas (cortex and thalamus) and in the circuit of remote control of seizures (substantia nigra pars reticulata). In order to determine whether protection of some brain areas could prevent the epileptogenesis induced by status epilepticus (SE) and to identify the cerebral structures involved in the genesis of TLE, we studied the effects of the chronic exposure to Vigabatrin (gamma-vinyl-GABA, GVG) on neuronal damage and epileptogenesis induced by lithium-pilocarpine SE. The animals were subjected to SE and GVG treatment (250 mg/kg) was initiated at 10 min after pilocarpine injection and maintained daily for 45 days. These pilo-GVG rats were compared with rats subjected to SE followed by a daily saline treatment (pilo-saline) and to control rats not subjected to SE (saline-saline). GVG treatment induced a marked, almost total neuroprotection in CA3, an efficient protection in CA1 and a moderate one in the hilus of the dentate gyrus while damage in the entorhinal cortex was slightly worsened by the treatment. All pilo-GVG and pilo-saline rats became epileptic after the same latency. Glutamic acid decarboxylase (GAD67) immunoreactivity was restored in pilo-GVG rats compared with pilo-saline rats in all areas of the hippocampus, while it was increased over control levels in the optical layer of the superior colliculus and the substantia nigra pars reticulata. Thus, the present data indicate that neuroprotection of principal cells in the Ammon's horn of the hippocampus is not sufficient to prevent epileptogenesis, suggesting that the hilus and extra-hippocampal structures, that were not protected in this study, may play a role in the genesis of spontaneous recurrent seizures in this model. Furthermore, the study performed in non-epileptic rats indicates that chronic treatment with a GABAmimetic drug upregulates the expression of the protein GAD67 in specific areas of the brain, independently from the seizures.

The lesional and epileptogenic consequences of lithium-pilocarpine-induced status epilepticus are affected by previous exposure to isolated seizures: effects of amygdala kindling and maximal electroshocks.

In temporal lobe epilepsy, the occurrence of seizures seems to correlate with the presence of lesions underlying the establishment of a hyperexcitable circuit. However, in the lithium-pilocarpine model of epilepsy, neuronal damage occurs both in the structures belonging to the circuit of initiation and maintenance of the seizures (forebrain limbic system) as in the propagation areas (cortex and thalamus) and in the circuit of remote control of seizures (substantia nigra pars reticulata). To determine whether or not we could protect the brain from lesions and epileptogenesis induced by status epilepticus and identify cerebral structures involved in the genesis of epilepsy, we studied the effects of the chronic exposure to non-deleterious seizures, either focalized with secondary generalization (amygdala kindling, kindled-pilocarpine rats), or primary generalized (ear-clip electroshocks, electroshock-pilocarpine rats) on neuronal damage and epileptogenesis induced by lithium-pilocarpine status epilepticus. These animals were compared to rats subjected to status epilepticus but not pretreated with seizures (sham-kindled-pilocarpine or sham-electroshock-pilocarpine rats). Compared to sham-pilocarpine rats, neuronal damage was prevented in the limbic system of the kindled-pilocarpine rats, except in the hilus of the dentate gyrus and the entorhinal cortex, while it was enhanced in rats pretreated with electroshocks, mainly in the entorhinal and perirhinal cortices. Most sham-kindled- and sham-electroshock-pilocarpine rats (92-100%) developed recurrent seizures after a silent period of 40-54days. Likewise, all kindled-pilocarpine rats developed spontaneous seizures after the same latency as their sham controls, while only two of 10 electroshock-pilocarpine rats became epileptic after a delay of 106-151days. The present data show that the apparent antiepileptic properties of electroshocks correlate with extensive damage in midbrain cortical regions, which may prevent the propagation of seizures from the hippocampus and inhibit their motor expression. Conversely, the extensive neuroprotection of the limbic system but not the hilus and entorhinal cortex provided by amygdala kindling does not prevent epileptogenesis. Thus, the hilus, the entorhinal and/or perirhinal cortex may be key structure(s) for the establishment of epilepsy.

Electroshocks delay seizures and subsequent epileptogenesis but do not prevent neuronal damage in the lithium-pilocarpine model of epilepsy.

Electroconvulsive therapy, which is used to treat refractory major depression in humans increases seizure threshold and decreases seizure duration. Moreover, the expression of brain derived neurotrophic factor induced by electroshocks (ECS) might protect hippocampal cells from death in patients suffering from depression. As temporal lobe epilepsy is linked to neuronal damage in the hippocampus, we tested the effect of repeated ECS on subsequent status epilepticus (SE) induced by lithium-pilocarpine and leading to cell death and temporal epilepsy in the rat. Eleven maximal ECS were applied via ear-clips to adult rats. The last one was applied 2 days before the induction of SE by lithium-pilocarpine. The rats were electroencephalographically recorded to study the SE characteristics. The rats treated with ECS before pilocarpine (ECS-pilo) developed partial limbic (score 2) and propagated seizures (score 5) with a longer latency than the rats that underwent SE alone (sham-pilo). Despite this delay in the initiation and propagation of the seizures, the same number of ECS- and sham-pilo rats developed SE with a similar characteristic pattern. The expression of c-Fos protein was down-regulated by repeated ECS in the amygdala and the cortex. In ECS-pilo rats, c-Fos expression was decreased in the piriform and entorhinal cortex and increased in the hilus of the dentate gyrus. Neuronal damage was identical in the forebrain areas of both groups, while it was worsened by ECS treatment in the substantia nigra pars reticulata, entorhinal and perirhinal cortices compared to sham-pilo rats. Finally, while 11 out of the 12 sham-pilo rats developed spontaneous recurrent seizures after a silent period of 40+/-27 days, only two out of the 10 ECS-pilo rats became epileptic, but after a prolonged latency of 106 and 151 days. One ECS-pilo rat developed electrographic infraclinical seizures and seven did not exhibit any seizures. Thus, the extensive neuronal damage occurring in the entorhinal and perirhinal cortices of the ECS-pilo rats seems to prevent the establishment of the hyperexcitable epileptic circuit.

C-Fos, Jun D and HSP72 immunoreactivity, and neuronal injury following lithium-pilocarpine induced status epilepticus in immature and adult rats.

In order to follow the maturation-related evolution of neuronal damage, cellular activation and stress response subsequent to Li-Pilo seizures in the 10- (P10), 21-day-old (P21) and adult rat, we analyzed the expression of the c-Fos protein as a marker of cellular activation, HSP72 immunoreactivity as the stress response and silver staining for the assessment of neuronal damage in 20 selected brain regions. The early wave of c-Fos measured at 2 h after the onset of seizures was present in most structures of the animals at the three ages studied and particularly strong in the cerebral cortex, hippocampus and amygdala. The late wave of c-Fos measured at 24 h after the onset of seizures and that was shown to correlate to neuronal damage was absent from the P10 rat brain, and present mainly in the cerebral cortex and hippocampus of P21 and adult rats. The expression of the stress response, assessed by the immunoreactivity of HSP72 at 24 h after the seizures was absent from the P10 rat brain and present in the entorhinal cortex, amygdala, hippocampus and thalamus of P21 and adult rats. The expression of Jun D at 24 h after the seizures was discrete and present in most brain regions at all ages. Neuronal injury assessed by silver staining at 6 h after the onset of seizures was very discrete in the brain of the P10 rat and limited to a few neurons in the piriform and entorhinal cortices. In older animals, marked neuronal degeneration occurred in the cerebral cortex, amygdala, hippocampus, lateral septum and thalamus. Thus the immediate cell activation induced by lithium-pilocarpine seizures which is present at all ages translates only into a late wave of c-Fos and the expression of HSP72 in P21 and adult animals in which there will be extensive cell damage.