Magnetic resonance diffusion tensor imaging for the pedunculopontine nucleus: proof of concept and histological correlation.

The pedunculopontine nucleus (PPN) has been proposed as target for deep brain stimulation (DBS) in patients with postural instability and gait disorders due to its involvement in muscle tonus adjustments and control of locomotion. However, it is a deep-seated brainstem nucleus without clear imaging or electrophysiological markers. Some studies suggested that diffusion tensor imaging (DTI) may help guiding electrode placement in the PPN by showing the surrounding fiber bundles, but none have provided a direct histological correlation. We investigated DTI fractional anisotropy (FA) maps from in vivo and in situ post-mortem magnetic resonance images (MRI) compared to histological evaluations for improving PPN targeting in humans. A post-mortem brain was scanned in a clinical 3T MR system in situ. Thereafter, the brain was processed with a special method ideally suited for cytoarchitectonic analyses. Also, nine volunteers had in vivo brain scanning using the same MRI protocol. Images from volunteers were compared to those obtained in the post-mortem study. FA values of the volunteers were obtained from PPN, inferior colliculus, cerebellar crossing fibers and medial lemniscus using histological data and atlas information. FA values in the PPN were significantly lower than in the surrounding white matter region and higher than in areas with predominantly gray matter. In Nissl-stained histologic sections, the PPN extended for more than 10 mm in the rostro-caudal axis being closely attached to the lateral parabrachial nucleus. Our DTI analyses and the spatial correlation with histological findings proposed a location for PPN that matched the position assigned to this nucleus in the literature. Coregistration of neuroimaging and cytoarchitectonic features can add value to help establishing functional architectonics of the PPN and facilitate neurosurgical targeting of this extended nucleus.

Basal dendrites are present in newly born dentate granule cells of young but not aged pilocarpine-treated chronic epileptic rats.

Epilepsy is known to influence hippocampal dentate granule cell (DGC) layer neurogenesis. In young adult rats, status epilepticus (SE) increases the number DGC newly borne cells and basal dendrites (BD), which persist at long-term. In contrast, little is known on whether these phenomena occur in elderly epileptic animals. In the present study, we compare DGC proliferation and the incidence of BD in young and aged pilocarpine-treated rats. Three epileptic groups were considered: Young animals given pilocarpine at 3 months of age. Aged animals treated with pilocarpine at 3 months of age that were sacrificed at 17-20 months. Aged animals that had pilocarpine and developed SE at 20 months, being sacrificed 2 months later. Nine days prior to sacrifice, animals underwent swimming sessions in the Morris water maze as a protocol for the development of hippocampal neurogenesis. We found a higher incidence of newly born DGC cells in young as compared to aged epileptic animals (P<0.001). This later group however, was not homogeneous. While a significant increase in DGC neurogenesis was observed when aged animals with long lasting epilepsy were compared to non-epileptic controls (P<0.01), this has not been recorded in aged animals that had epilepsy for only 2 months (P>0.05). When the number of DGC containing BD was considered, a significantly higher incidence was observed in young as compared to aged epileptic rats (P=0.001). Animals in this later group virtually lacked BD in newly formed dentate gyrus (DG) cells. Based on these results we conclude that plastic changes during epileptogenesis and the development of a pathological substrate in young animals is associated with DGC proliferation and the emergence of BD. As aging occurs, DGC neurogenesis can still be induced in rats with a long-term history of epilepsy but the emergence of BD is markedly reduced.

Neo-Timm staining in the thalamus of chronically epileptic rats.

The thalamus is an important modulator of seizures and is severely affected in cholinergic models of epilepsy. In the present study, chronically epileptic rats had their brains processed for neo-Timm and acetylcholinesterase two months after the induction of status epilepticus with pilocarpine. Both controls and pilocarpine-treated animals presented neo-Timm staining in the anterodorsal nucleus, laterodorsal nucleus, reticular nucleus, most intralaminar nuclei, nucleus reuniens, and rhomboid nucleus of the thalamus, as well as in the zona incerta. The intensity of neo-Timm staining was similar in control and pilocarpine-treated rats, except for the nucleus reuniens and the rhomboid nucleus, which had a lower intensity of staining in the epileptic group. In animal models of temporal lobe epilepsy, zinc seems to modulate glutamate release and to decrease seizure activity. In this context, a reduction of neo-Timm-stained terminals in the midline thalamus could ultimately result in an increased excitatory activity, not only within its related nuclei, but also in anatomical structures that receive their efferent connections. This might contribute to the pathological substrate observed in chronic pilocarpine-treated epileptic animals.

Neo-Timm staining in the thalamus of chronically epileptic rats

The thalamus is an important modulator of seizures and is severely affected in cholinergic models of epilepsy. In the present study, chronically epileptic rats had their brains processed for neo-Timm and acetylcholinesterase two months after the induction of status epilepticus with pilocarpine. Both controls and pilocarpine-treated animals presented neo-Timm staining in the anterodorsal nucleus, laterodorsal nucleus, reticular nucleus, most intralaminar nuclei, nucleus reuniens, and rhomboid nucleus of the thalamus, as well as in the zona incerta. The intensity of neo-Timm staining was similar in control and pilocarpine-treated rats, except for the nucleus reuniens and the rhomboid nucleus, which had a lower intensity of staining in the epileptic group. In animal models of temporal lobe epilepsy, zinc seems to modulate glutamate release and to decrease seizure activity. In this context, a reduction of neo-Timm-stained terminals in the midline thalamus could ultimately result in an increased excitatory activity, not only within its related nuclei, but also in anatomical structures that receive their efferent connections. This might contribute to the pathological substrate observed in chronic pilocarpine-treated epileptic animals.

Spontaneous subarachnoid hemorrhage as the primary manifestation of carotid cavernous fistulas: case report.

We report the case of a 19-year old male patient initially admitted to our service after a motor vehicle accident with a normal neurologic evaluation and a CT scan that revealed no abnormalities. Nineteen months later, he was readmitted after a subtle headache episode, followed by a brief loss of consciousness. He was submitted to a complete evaluation, which revealed no abnormalities (even in the neurologic and ophthalmologic exams). A CT was performed revealing a diffuse subarachnoid hemorrhage. Contrast enhancement displayed a right paraselar lesion, which was first interpreted as a giant aneurysm. The patient underwent a cerebral angiography which showed a right carotid-cavernous fistula with retrograde venous drainage through the superior and inferior petrosal sinuses. Filling of various cortical vessels was observed. The patient was treated with endovascular technique and a control angiographic study assured the complete closure of the fistula. He had an excellent clinical recovery, being discharged in good conditions.

Loss of NADPH diaphorase-positive neurons in the hippocampal formation of chronic pilocarpine-epileptic rats.

Recent evidence suggests an important role for NO in cholinergic models of epilepsy. Nicotinamide adenine dinucleotide phosphate diaphorase (NADPHd), a marker of NO containing neurons, was shown to intensely colocalize with GABA in double-labeling studies performed in the hippocampal formation (exception made for the pyramidal cell layer) (Valtschanoff et al., J Comp Neurol 1993:331:111-121). In this sense, it further characterizes an extremely important cell category due to the relevant involvement of inhibitory systems in the mechanisms of genesis and propagation of seizures. Here, we assessed the histochemistry for NADPHd in the hippocampal complex of chronic pilocarpine-epileptic animals. NADPHd-positive cells were lost in almost every hippocampal subfield in pilocarpine-treated rats. The central portion of the polymorphic layer of the dentate gyrus (hilus) presented one of the highest losses of NADPHd-positive cells (55-79%) in the hippocampus. A significant loss of NADPHd-positive cells was seen in strata oriens, pyramidale, and radiatum CA1, CA2, and CA3 subfields. NADPHd staining in the subicular pyramidal cell layer was not different from that observed in controls. A significant loss of NADPHd-stained cells was observed in entorhinal cortex layers II and III in the epileptic group. For entorhinal cortex layers V and VI, however, results varied from an almost complete tissue destruction to an overexpression of NADPHd-positive cells, as well as an increase in neuropil staining. In summary, loss of NADPHd staining was not uniform throughout the hippocampal formation. There has been a growing support for the notion that GABAergic neurons in the hippocampal formation are not equally sensitive to insults. Our results suggest that, as a marker for a subpopulation of GABAergic neurons, NADPHd helps in further refining the characterization of the different neuronal populations sensitive to epileptic activity.

Status epilepticus induced by pilocarpine and picrotoxin.

Since its original description over 10 years ago, the pilocarpine model of status epilepticus (SE) has gained considerable attention. Much work has been done with the model in order to characterize the involvement of different brain structures in seizure genesis and spread. Electrophysiological studies of temporal lobe epileptic slices of both human and animal models, have failed to reveal hyperexcitability, unless blockade of GABAergic inhibition is performed. Thus, we have decided to evaluate potential contributions of picrotoxin, a GABAA channel blocker, on pilocarpine-induced SE. Animals injected with three-specific dose combinations (pilocarpine dose/picrotoxin dose), 150/0.5, 75/1.5 and 50/2.0 mg/kg, evoked status epilepticus (SE) within 23, 31 and 27 min, respectively. Ictal events and EEG spikes were initially observed either in the amygdala or in the hippocampus, with a later spread to cerebral cortex. Neuropathological analysis, performed 5-7 days after SE, has shown a high degree of cell loss predominantly in the piriform cortex, amygdala, hippocampus, thalamus and substantia nigra. Mortality rates for 150/0.5, 75/1.5 and 50/2.0 mg/kg (pilocarpine dose/picrotoxin dose) were 53, 42 and 51%, respectively. Single injections of 150 mg/kg of pilocarpine or 3 mg/kg of picrotoxin did not evoke any form of sustained epileptic activity. Previous studies in which simultaneous injections of other GABAA antagonists (i.e. bicuculline) and pilocarpine were performed, did not show clear evidences of a synergistic action between these two systems. The present study reveals a proconvulsant role for picrotoxin when co-administered with subconvulsant doses of pilocarpine. Possible mechanisms that might account for the interactions between the cholinergic and GABAergic systems in regard to epileptogenesis are discussed.