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Alumina gel injections into the temporal lobe of rhesus monkeys were used in an attempt to mimic complex partial seizures associated with temporal lobe epilepsy in humans (Ribak et al., 1998). Our analysis is based on injections of alumina gel into several regions of the temporal lobe in rhesus monkeys. Thus structures that were injected include the hippocampus, entorhinal and perirhinal cortices, and the amygdala. In all cases, complex partial and secondarily generalized seizures were observed. They had many similarities to the ictal symptoms of human temporal lobe epilepsy.

Methods of Generation

Adult or adolescent monkeys are placed under general anesthesia, and stereotactic injections are made into the mesial temporal structures. In the past these injections have been performed through an approach from above that enters the ventricle. The disadvantage of that approach is that the alumina gel can back-track into the ventricles and spread beyond the desired location. With an updated lateral approach, the tract is below the ventricle so the needle can approach the amygdala, hippocampus, or entorhinal cortex (Ribak et al., 1998). This approach requires a specially designed right-angle needle of approximately 23-gauge (higher gauges tend to deflect in variable directions as a result of the lack of rigidity). The musculus temporalis must be split and the zygoma removed along the path to the target. A small craniotomy is made in the temporal bone, and the dura opened along the needle trajectory. Targets have been selected in the past with stereotactic atlases. However, with current imaging technology, the monkey can be placed in a magnetic resonance imaging (MRI)-compatible stereotactic frame, and the needle directed to its target based on the anatomy of the individual animal. Injections consist of 0.1 to 0.2 ml of alumina gel. The most reliable seizure activity occurs with injections into the entorhinal cortex, 2 to 3 weeks after injection. With hippocampal or amygdala injections, sometimes the seizures evolve so rapidly that after the first several seizures a secondary generalization occurs. A high rate of seizure frequency can endanger the monkey's health. A somewhat slower, more stable pattern is generally observed with entorhinal injections or mesial temporal cortical injections. Injected subjects develop electrophysiologic and behavioral characteristics of complex partial seizures. Chronic spontaneous seizures have been observed with this technique, and electrographic seizures can be recorded using epidural electrode strips (Figure 3).

Characteristics and Defining Features Behavioral and Clinical Features

Alumina gel injections into the hippocampus result in clinical seizure activity that typically develops 12 to 14 days after the injections (Ribak et al., 1998). This seizure activity is characterized as complex partial seizures with secondary generalization. Alumina gel injections into the

FIGURE 3 X-rays of the anterior-posterior (A) and lateral (B) views of a monkey implanted with epidural electrodes. Lead 1 is in the mesial-inferior temporal region, lead 2 is over the inferior temporal region, lead 3 is over the superior temporal region, and lead 4 is over the parietal cortex. The electrical connections are buried subcuticularly and emerge through a superior head plug (arrows). (Reprinted with permission from Ribak et al., 1998.)

FIGURE 3 X-rays of the anterior-posterior (A) and lateral (B) views of a monkey implanted with epidural electrodes. Lead 1 is in the mesial-inferior temporal region, lead 2 is over the inferior temporal region, lead 3 is over the superior temporal region, and lead 4 is over the parietal cortex. The electrical connections are buried subcuticularly and emerge through a superior head plug (arrows). (Reprinted with permission from Ribak et al., 1998.)

amygdala develop more slowly, typically over a 3- to 6-week period (Ribak et al., 1998). Complex partial seizures result from these injections, and they persist over a long period. Alumina gel injections into entorhinal and perirhinal cortices result in similarly characterized complex partial seizures within 2 to 3 weeks (Ribak et al., 1998). Overall, alumina gel injections into mesial temporal lobe structures of monkeys result in subclinical, complex partial and secondarily generalized seizures that have many similarities to the ictal symptoms of human temporal lobe epilepsy (Sharbrough, 1987). Injections of the inferior temporal gyrus showed no behavioral abnormalities and served as a control for the other injected regions of the temporal lobe that are responsible for causing complex partial seizures (Ribak et al., 1998).

When complex partial seizures developed in these monkeys, the behavioral pattern was remarkably similar (Ribak et al., 1998). In all instances the seizures started with blank stares followed by head turning. In monkeys injected into the hippocampus, this behavior rapidly became secondarily generalized. Other symptoms included smelling of the fingers, followed by an arrest of movement with a motionless stare for up to a minute. As the seizure generalized, these behaviors evolved to include vocalization, drooling, open-mouth oral-facial automatisms, chewing motions, and head turning with extremity automatisms. Infrequently the secondary generalization would lead to tonic-clonic Jacksonian march.

Electrographic and Electroencephalograph^ Features

Electroencephalography can be used to localize the origin of abnormal activity before seizures. Alumina gel injections in the amygdala, perirhinal and entorhinal cortices, or

Ammon's horn and dentate gyrus all initially displayed focal pathological EEG slowing limited to the injection site. After clinical seizures developed, widespread pathological EEG slowing over both hemispheres followed. In addition, the monkeys displayed the cardinal ictal and interictal epilepti-form EEG abnormalities limited to the mesial-inferior temporal lobe on the side ipsilateral to the injection site (Figure 4). Furthermore, other ipsilateral and contralateral structures were observed to have different degrees of spread. These electrophysiologic abnormalities are very similar to those observed in human temporal lobe epilepsy (Ribak et al., 1998).

Neuropathology

Cell Loss

Blocks of tissue from the temporal lobe and homologous area in the contralateral nonepileptic temporal lobe were analyzed in light and electron microscopic preparations (Ribak et al., 1998). Nissl-stained sections were used to determine the full extent of the alumina gel injection sites into the different regions of the temporal lobe. Similar to results in the sensorimotor cortex, neurons were lost at injections sites. In addition, one injection site was associated with distant cell loss (Ribak et al., 1998). Thus alumina gel injections into the amygdala were accompanied by cell loss in the CA1 region of the hippocampus, hilar region of the dentate gyrus, and layer III of entorhinal and perirhinal cortex.

Reactive Gliosis

Increased numbers of glial cells occurred in various regions of the temporal lobe, where cell loss occurred following injections (Ribak et al., 1998). The increased gliosis

FIGURE 4 Interictal EEG activites (during the first 5 seconds of this epoch) demonstrate asynchronous pathological delta slowing over both hemispheres, with greater delta slowing over the more medial left-sided electrodes. Fast activities (beta and alpha activities) are consistently of higher amplitude over the right side (contralateral to alumina gel injection) interictally. The recording instrument was turned off for about 5 seconds for pen adjustment (indicated by a gap after the interictal sample), and the electrographic seizure had begun when recording resumed. The electrographic seizure consisted initially of high amplitude, shaply contoured, rhythmic theta activities at LtSDl with lower amplitude representation at the adjacent LtSD2 electrode, and with irregular slowing at other sites; at this time the animal responded to voice by orienting its head and gaze. Twelve seconds later, the ictal discharge had evolved into bursting polyspike activities and had spread to involve LtSD3 and LtSDl-2. By this time, the contralateral electrodes (RtSDl-4) are recording ictal activities of predominantly 2.5-3Hz rhythmic slowing with superimposed spikes. The animal failed to respond to voice (Call) and gradually assumed a fixed posture of rightward ocular, cephalic, and truncal version. This posturing was maintained throughout the remainder of the electrographic seizure, which lasted 110 sec. The electro-graphic seizure did not spread to suprasylvian electrodes and the animal did not convulse. The recording was performed using published parameters (Ribak et al. 1998). Note: The wire of the right suprasylvian subdural electrode (RtSD4) was broken, so this channel was turned off during the recording. LtSD, left subdural contacts; RtSD, right subdural contacts; Al, left ear; A2, right ear. Reprinted with permission from Ribak et al. (1998).

was also observed in the molecular layer of the dentate gyrus. Electron microscopy confirmed the presence of reactive astrocytes in the hippocampus and dentate gyrus (Ribak et al., 1998).

Plasticity and Mossy Fiber Sprouting

In Timm-stained light microscopic preparations, the dentate granule cells exhibited aberrant mossy fiber sprouting into the inner molecular layer (Ribak et al. 1998). Electron microscopy showed that many of these mossy fibers formed synapses with the proximal dendrites and dendritic spines of granule cells (Figure 5). The presence of these mossy fibers on identified granule cells indicates increased recurrent excitatory circuitry in the brains from these monkeys.

Light microscopy also showed dispersion of the granule cell layer (Ribak et al., 1998). Thus the granule cells were radially organized (Figure 6). Electron microscopy of these orthogonal rows of granule cells showed intervening radially oriented processes of reactive astrocytes. These processes arose from hypertrophied astrocytes in the subgranular zone (Ribak et al., 1998). Thus some of the morphologic features observed following injections of alumina gel into temporal lobe structures are similar to the neuropathologic changes found in human temporal lobe epilepsy.

Limitations

Development of complex partial seizure activity is reasonably reliable to produce. The seizure pattern is frequently extremely stereotypic, but it requires electrophysiologic monitoring because the complex partial seizure may be easily misinterpreted as normal monkey behavior. There is high variability in the rate of seizure activity, and this variability tends to be site-specific. Seizures often develop with such severity that long-term studies are not possible. The temporal lobe alumina gel injection most likely models a mesial temporal lesion, but how effectively it mimics mesial temporal sclerosis remains to be determined. Nevertheless, it is a method of developing mesial temporal epilepsy in nonhuman primates that are anatomically far closer to human hippocampal structure than is the rat. The disadvantages of the model are similar to those of the sensorimotor focal epilepsy model. In addition, however, our experience is extremely limited with this model. Nevertheless it offers great promise for the future and needs to be evaluated further.

Insights into Human Disorders

This temporal lobe epilepsy model in monkeys has the potential to provide useful information about the patho-

FIGURE 5 Electron micrographs of a granule cell obtained from the dentate gyrus ipsilateral to the amygdala injected with alumina gel. A: The nucleus (N) and proximal apical dendrite (d) of the granule cell. Apposed to the ruffled surface of the dendrite is a large axon terminal that has the features of a mossy fiber. Also note the processes of astrocytes (as) with glial filaments. B: An enlargement of the mossy fiber in (A) showing that this mossy fiber forms asymmetrical synapses (arrows) with the proximal dendrite. A spine (s) appears within this mossy fiber. Scale bars = 1 mm. (Reprinted with permission from Ribak et al., 1998.)

FIGURE 5 Electron micrographs of a granule cell obtained from the dentate gyrus ipsilateral to the amygdala injected with alumina gel. A: The nucleus (N) and proximal apical dendrite (d) of the granule cell. Apposed to the ruffled surface of the dendrite is a large axon terminal that has the features of a mossy fiber. Also note the processes of astrocytes (as) with glial filaments. B: An enlargement of the mossy fiber in (A) showing that this mossy fiber forms asymmetrical synapses (arrows) with the proximal dendrite. A spine (s) appears within this mossy fiber. Scale bars = 1 mm. (Reprinted with permission from Ribak et al., 1998.)

physiologic mechanism of human temporal lobe epilepsy. It is well known that chronic seizures may cause hippocampal neuronal loss and behavioral changes in human temporal lobe epilepsy. Serial chronologic studies of this alumina gel model in monkeys might address this question and others. For example, demonstration of interictal disturbances of learning and other behaviors in this model could be used to develop therapies for the interictal impairments that occur in human temporal lobe epilepsy. Also, this model may be useful in testing new medical and surgical therapies for complex partial seizures in humans. This monkey model of temporal lobe epilepsy has the potential to provide valuable information about the cellular mechanisms of temporal lobe epilepsy in humans because it is more like human temporal lobe epilepsy than existing rodent models.

Acknowledgments

The authors gratefully acknowledge the technical assistance of previous technicians and the cooperation of previous collaborators. This work was supported by the Medical Research Service of the Department of Veterans Affairs, the Yerkes Regional Primate Research Center, National Institutes of Health Core Grant RR-00165 (R.A.E.B), National Institutes of Health Grants NS-15669 and NS-38331 (C.E.R.), and National Institutes of Health Training Grant T32-NS045540 (supporting L.A.S.).

FIGURE 6 Photomicrographs of Nissl-stained sections through the hippocampus from the control hemisphere (A) and the amygdala injected (B) side of a monkey. In (A) the granule cell layer (G) shows a normal appearance. In contrast, the granule cell layer (G) in B is dispersed. Scale bar = 200 mm. (Reprinted with permission from Ribak et al., 1998.)

FIGURE 6 Photomicrographs of Nissl-stained sections through the hippocampus from the control hemisphere (A) and the amygdala injected (B) side of a monkey. In (A) the granule cell layer (G) shows a normal appearance. In contrast, the granule cell layer (G) in B is dispersed. Scale bar = 200 mm. (Reprinted with permission from Ribak et al., 1998.)

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