Methods Of Preparation And Generation Of Epileptiform Discharges

The methods used to isolate and maintain in vitro a guinea pig brain have been extensively described (de Curtis et al., 1991, 1998; Llinas et al., 1981, 1989; Muhlethaler et al., 1993). Young adult guinea pigs weighing 150 to 250g are anesthetized with 70mg/kg tiopenthal, administered intraperioneally. A study of the time course of barbiturate washout in this preparation demonstrated that within 1 hour from the in vitro isolation, the brain concentration of the anesthetic measured by high-performance liquid chromatography (HPLC) is reduced to values close to zero (Librizzi et al., unpublished observations), which exclude possible pharmacologic interference with the recorded activity. After exposing the heart, intracardiac perfusion with a cold saline solution (see later discussion) at 14° C is performed to reduce brain metabolism and to preserve the brain tissue during the dissection. The animal is decapitated after 3 minutes, and the brain is carefully isolated and transferred to the incubation chamber. The surgical maneuver to isolate the brain does not differ substantially from the technique used to prepare brain slices, but it must be performed rapidly (i.e., in 6 to 8 minutes). The details are reported in Muhlethaler et al. (1993). After dissection the isolated brain is positioned in the incubation chamber with its ventral surface upward to visualize the base of the brain and the vascular system formed by the basilar artery and the circle of Willis that is removed en bloc with the brain. The isolated brain is held down by two silk threads secured to the bottom of the incubation chamber to improve mechanical stability. The dura that enfolds the basilar artery is removed, a cannula (PE 60 tubing tapered to about 300-|mm-tip diameter) is inserted into the basilar artery, and the cannula is secured by tying a thin silk thread around the artery. Arterial perfusion at a rate of 5.5ml per minute is restored with the following solution: NaCl 126mM, KCl 2.3mM, NaHCO3 26mM, MgSO4 1.3mM, CaCl2 2.4mM, KH2PO4 1.2mM, glucose 15mM, 4-(2-hydroxyethyl)-1-piperazine-ethanesulfonic acid (HEPES) 5mM, and 3% dextran 70.000 (pH 7.3) saturated with a 95 to 5% O2-CO2 gas mixture. The same solution is used for the intracardiac perfusion with a slightly acidic pH (7.1) to enhance protection of the tissue during dissection. In the perfusion chamber, the hypophyseal and carotid arteries are ligated with silk knots. The temperature of the incubation chamber is slowly increased to 32° C (0.2°C per minute). The arterial pressure of the isolated brain, as measured by a pressure transducer interposed along the perfusion line, ranges between 45 and 65 mmHg.

Brains are commonly isolated from young adult guinea pigs, 15 days to 4 months of age. No attempts have been performed to date to isolate the brains of younger and older animals. Viable isolated guinea pig brains can be reliably reproduced by expert experimenters. Since setting up the method at the Department of Experimental Neurophysiology of the Istituto Nazionale Neurologico, more than 10 young scientists have been trained to isolate guinea pig brains; all have mastered the isolation technique within 2 to 3 months.

Brains can be isolated in vitro from species such as guinea pigs that show a peculiar arrangement of the communication between the posterior (vertebrobasilar) and the anterior (carotid) arterial systems that form the circle of Willis. The basilar artery in the guinea pig divides into two large-diameter posterior communicating arteries (Figure 1), from which the posterior cerebral arteries originate. The guinea pig is the only animal analyzed so far in which the presence of these large-capacity posterior communicating arteries and the arrangement of the circle of Willis are compatible with perfusion of the entire brain via the basilar artery. In other animal species, such as the rat and the mouse (see Figure 1A), the small diameter of the posterior communicating arteries does not allow good brain perfusion in vitro when the basilar artery is cannulated. No attempts have been made to evaluate the feasibility of the brain isolation technique in animal species other than the rat, the mouse, and the guinea pig.

An acute epileptogenic condition can be easily and reliably established in the isolated guinea pig brain preparation. Interictal and ictal discharges can be consistently reproduced by brief arterial applications of proconvulsive compounds that are permeable to the blood-brain barrier. Three-minute applications of bicuculline (50 ||M), penicillin (1000 units/ml), or picrotoxin (1mM) diluted in the perfusion solution induce interictal spikes in the piriform-entorhinal cortex; these applications are followed, within 5 to 10 minutes, by ictal discharges that typically originate in the

FIGURE 1 A: Schematic representation of the circle of Willis of the rat (left) and the guinea pig (right). 1, Anterior communicating artery; 2, anterior cerebral artery; 3, middle cerebral artery; 4, carotid artery; 5, hypophyseal artery; 6, posterior communicating artery; 7, posterior cerebral artery; 8, Superior cerebellar artery; 9, basilar artery. B: Arteriography image obtained by perfusing an isolated guinea pig brain with 0.2 ml iodine contrast medium in the basilar artery. Large arteries of the circle of Willis are shown during an early perfusion time of the contrast medium.

rat guinea pig

FIGURE 1 A: Schematic representation of the circle of Willis of the rat (left) and the guinea pig (right). 1, Anterior communicating artery; 2, anterior cerebral artery; 3, middle cerebral artery; 4, carotid artery; 5, hypophyseal artery; 6, posterior communicating artery; 7, posterior cerebral artery; 8, Superior cerebellar artery; 9, basilar artery. B: Arteriography image obtained by perfusing an isolated guinea pig brain with 0.2 ml iodine contrast medium in the basilar artery. Large arteries of the circle of Willis are shown during an early perfusion time of the contrast medium.

hippocampus-entorhinal cortex and secondarily invade the perirhinal cortex (Figure 2). Unlike results reported for cats in vivo (Avoli and Gloor, 1982; Gloor et al., 1977), GABAergic antagonists in the isolated guinea pig brain preparation induce epileptiform discharges that demonstrate a clear focal onset in the mesial temporal lobe. Typical ictal events are characterized by fast activity (around 20 to 25 Hz) that originates from the hippocampus-entorhinal cortex and builds up in 2 or 3 seconds (Figure 2b) superimposed on a slow extracellular voltage shift. The fast activity is followed by phasic runs of high-amplitude afterdischarges (0.5 to 1 second in duration) widely distributed in the hippocampus and parahippocampal regions (Figure 2c) that progressively become more regular and increase in amplitude. Afterdischarge can propagate to regions in which the early part of the seizure was never observed, such as the piriform cortex or the neocortex. After 2 to 4 minutes, afterdischarges decrease in duration and periodicity and ultimately disappear. A postictal depression of several tenths of minutes follows, during which epileptiform discharges could not be induced, but afferent stimulation can evoke responses in the limbic region. Ictal epileptiform discharges repeat for several minutes or hours after a single arterial perfusion with GABA-receptor antagonists. A normal excitability condition resumes within 2 to 3 hours after bicuculline application.

Ictal activity generated in the temporolimbic region does not propagate diffusely into the brain; for instance, ictal epileptiform discharges were never observed in the neocor-tex and seldom occurred in the piriform cortex (Librizzi and de Curtis, 2003; Uva et al., unpublished observations). For this reason the experimental procedure can be proposed as a model of acute focal epileptogenesis of the temporal lobe.

The ictal pattern generated in the isolated brain preparation is similar to that commonly observed during stereo electroencephalograph^ (EEG) recordings from human epileptogenic areas (Francione et al., 1994, 2003; Lieb et al., 1976; Pacia and Ebersole, 1999; Spencer and Spencer 1994; Tassi et al., 2002; Towsend and Engel, 1991; Wendling et al., 2002; Young Jung et al., 1999) characterized by the appearance of fast activity at the onset, followed by afterdischarges. Such an ictal onset differs from the pattern usually induced by the application of epilepto-genic drugs in slices. In most acute pharmacologic models developed in slices of hippocampus or cortex, ictal-like events are formed by large-amplitude afterdischarges characterized by repeated, fast-onset paroxysmal depolarizing shifts (PDSs) that gradually decline in frequency to about 2 to 10 Hz, interpreted as a preictal event or subclinical "embryo seizure" (Ralston, 1958). The differences between the pattern observed in the isolated guinea pig brain and that observed in slices probably depends on the restricted connectivity preservation in brain tissue slices.

Muscarinic receptor agonists (carbachol, pilocarpine) do not induce epileptiform activity unless they are co-perfused with a GABA-receptor antagonist. Unlike the case for cortical slices (Nagao et al., 1996), arterial perfusion with pilocarpine alone at concentrations between 10 and 100 ||M, for periods up to 2 hours, never resulted in epileptiform discharges, even though intraperitoneal application of pilocarpine in the guinea pig in vivo induced a prolonged status epilepticus.

FIGURE 2 Representative ictal discharge recorded in the olfactory and limbic cortices of the isolated guinea pig brain preparation after arterial perfusion of 50 ||M bicuculline. Recordings were performed in the piriform cortex (PC), in the entorhinal cortex (EC), in the CA1 region of the hippocampus (hip), and in the perirhinal cortex (PRC), as illustrated on the ventral view of the guinea pig brain on top. Expanded traces in a, b, c, and d are sampled from the upper traces. The ictal onset of the discharge features fast activity that originates in the hippocampus. For details see text.

FIGURE 2 Representative ictal discharge recorded in the olfactory and limbic cortices of the isolated guinea pig brain preparation after arterial perfusion of 50 ||M bicuculline. Recordings were performed in the piriform cortex (PC), in the entorhinal cortex (EC), in the CA1 region of the hippocampus (hip), and in the perirhinal cortex (PRC), as illustrated on the ventral view of the guinea pig brain on top. Expanded traces in a, b, c, and d are sampled from the upper traces. The ictal onset of the discharge features fast activity that originates in the hippocampus. For details see text.

Focal hypersynchronous potentials can be reliably induced by local application of proconvulsive compounds. Intraparenchymal injection of bicuculline (1 to 2mM) or picrotoxin (10mM) in the piriform cortex induces large-amplitude interictal spikes that repeat with a 5- to 10-second periodicity and propagate to synaptically connected regions, such as the amygdala, the periamygdaloid cortex, and the lateral entorhinal cortex (Biella et al., 1996, 2003). Unlike systemic applications, local drug treatment in the piriform cortex did not induce ictal discharges but could promote the generation of brief afterdischarges. To date no attempts have been performed to trigger epileptiform activity by local applications of drugs in structures other than the piriform region.

An alternative procedure that has been used to generate epileptiform discharges in the hippocampal-parahippocam-pal area is represented by direct application of tetanic stimulation to the cerebral tissue at 100 Hz for 1 second, as reported by Paré and colleagues (1992). When such prolonged tetanic stimuli are repeated for three to five cycles at 0.5 Hz, self-sustained epileptiform afterdischarges of brief duration are generated that only occasionally develop into seizure-like activity with features similar to those described above for the pharmacologic model.

ADVANTAGES, LIMITATIONS, AND FUTURE DEVELOPMENTS

The advantages of the isolated guinea pig brain with respect to other in vitro and the in vivo conditions and preparations have been discussed extensively in previous articles (de Curtis et al., 1991; Llinas et al., 1981; Muhlethaler et al., 1993). The most obvious advantage is preservation of the tridimensional connections between close and remote brain areas, which allows evaluation of the unrestricted expres sion of network interactions during epileptiform discharges. Moreover, use of the in vitro brain preparation allows (1) approaching brain regions that are otherwise difficult to access in vivo; (2) evaluating the tangential propagation of activity across the surface of the brain with a more direct and facilitated approach than in vivo by means of electro-physiologic recordings and optical imaging; (3) performing multisite recordings at extracellular and intracellular levels; (4) performing pharmacologic tests by perfusing drugs via the arterial system in a close-to-in vivo situation in which the blood-brain barrier is functionally active; (5) applying drugs that cannot be used in vivo because of their severe, if not lethal, systemic effects.

Finally, because of the in situ preservation of the vascular system in the isolated guinea pig brain, the mechanisms of interactions between neuronal activity and the vascular compartment can be analyzed. It is well known that bidirectional neurovascular-neuronal interactions regulate brain excitability. Neuronal and glial activities are coupled to localized changes in cerebral blood flow that rely on several mechanisms, such as changes in local concentration of ions and the release of classic neurotransmitters or neuromodu-lators (e.g., nitric oxide or adenosine). Further changes in blood flow and in blood-brain barrier permeability influence metabolism and activity of the brain, which result in dramatic excitability changes (Akgoren et al., 1996; Gaillard et al., 1995; Logothetis et al., 2001; Magistretti et al., 1999; Iadecola et al., 1997; Malonek and Grinvald, 1996; Villringer and Dirnagl, 1995; Zonta et al., 2003). The neuronal and vascular compartments during epileptiform activation have been studied simultaneously in the past during pioneer studies in vivo (Caspers and Speckmann, 1972; Jasper and Erickson, 1941; Paulson and Sharbrough, 1974; Penfield et al., 1940) that have been largely overlooked for several decades in epilepsy research field in favor of the study of intrinsic excitability properties and synaptic interactions between neurons. With the spread of diagnostic imaging technology, such as single-photon emission computed tomography (SPECT), positron emission tomography (PET), and functional magnetic resonance imaging (MRI), the interactions between blood flow, brain metabolism, and neuronal activity have been reconsidered (Arthurs and Boniface, 2002; Duncan, 1992; Logothetis et al., 2001). Very preliminary attempts to correlate brain activity with changes in blood flow during an ictal epileptiform discharge have been carried out on the isolated brain preparation (de Curtis et al., 1998). As illustrated in Figure 3, the time course of seizure-like events induced by arterial application of bicu-culline can be correlated with the simultaneous changes in extracellular ion concentrations (potassium and protons/pH) and changes in resistance to blood flow, measured with a pressure transducer positioned along the perfusion line. These studies could be further detailed by measuring changes in the size of pial vessels with a video microscopy

FIGURE 3 Simultaneous recording of ictal discharges elicited by arterial application of penicillin (1000 units/ml). Two-barrel electrodes recorded field potentials (FP) simultaneously with extracellular potassium ([K+]o) and proton (pH) concentration in the extracellular space by means of ion-selective electrodes. The changes in brain blood flow associated with seizure activity were simultaneously measured as changes in the resistance to flow by a pressure transducer inserted along the perfusion line just upstream of the insertion of the polyethylene cannula in the basilar artery. VT, vascular tone. Downward deflections represent vasodilation associated with an increase in blood flow.

FIGURE 3 Simultaneous recording of ictal discharges elicited by arterial application of penicillin (1000 units/ml). Two-barrel electrodes recorded field potentials (FP) simultaneously with extracellular potassium ([K+]o) and proton (pH) concentration in the extracellular space by means of ion-selective electrodes. The changes in brain blood flow associated with seizure activity were simultaneously measured as changes in the resistance to flow by a pressure transducer inserted along the perfusion line just upstream of the insertion of the polyethylene cannula in the basilar artery. VT, vascular tone. Downward deflections represent vasodilation associated with an increase in blood flow.

system to evaluate changes in local cerebral blood flow during brain activity.

Even though a detailed neuropathologic study of isolated brains after the induction of repetitive seizures has never been performed, no obvious damage was observed in the ictal onset region (hippocampus and entorhinal cortex) after thionine staining of the tissue (performed to identify the position of the recording and stimulating electrodes at the end of the electrophysiologic experiment). Histochemical studies of the isolated brains after induction of epileptiform discharges could be performed, in principle, to analyze acute changes in brain tissue, such as induction of immediate early genes, inflammatory molecules, edema, etc. In addition, MRI scans of in vitro isolated or postfixed guinea pig brains (Figure 4) can be performed with a spatial resolution that allows identification of an altered signal in the gray and white matter, such as postepileptic brain edema or alterations associated with sclerosis or gliosis (not shown).

The main limitation of the currently available technique is that it models an acute epileptogenic condition. Future developments include the possibility of isolating brains from animals in which a chronic epileptic condition has been established. Preliminary experiments on the pilocarpine model have been performed (Turski et al., 1989). Intraperitoneal injection of 380 ||M of pilocarpine induces in guinea pigs subcontinuous partial and secondary generalized seizures lasting several hours this activity can be effectivey terminated by intraperitoneal injection of benzodiazepines. Brains isolated just after the epileptic status produce

Chapter 9/In Vitro Isolated Guinea Pig Brain

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FIGURE 4 High-definition magnetic resonance T2-weighted images of the isolated brain of the guinea pig performed with a 1.5 Tesla Philips instrument (courtesy of Dr. Maria Grisoli and Dr. Maria Grazia Bruzzone).

spontaneous epileptiform activity without further pharmacologic or stimulation procedures. Ideally brain isolation could be performed at different times during the "latent period" as well as before and after establishment of a chronic epileptic condition. The use of an in vitro isolated brain for such a study would allow precise reconstruction of the patterns of generation and propagation of epileptiform discharges using microphysiologic and imaging techniques, under conditions that preserve the tridimensional connectivity among brain structures.

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