Lidocaine Suppresses the Anoxic Depolarization and Reduces the Increase in the Intracellular Ca sup 2+ Concentration in Gerbil Hippocampal Neurons 

The study was approved by the Committee on Animal Experimentation at Ehime University School of Medicine, Ehime, Japan. Male Mongolian gerbils weighing 60–80 g (Seiwa Experimental Animals, Fukuoka, Japan) were housed in groups in a room controlled at 23 +/- 1 [degree sign] Celsius and maintained in an alternating 12-h light and 12-h dark cycle (lights on at 6:00 A.M.). Animals were deprived of food but not water at least 6 h before ischemia because of the influence of hyperglycemia on ischemic brain damage. [9] All experiments were performed under spontaneous ventilation. In experiment 1, the changes in the DC potential shift produced by transient forebrain ischemia in the hippocampal CA1 field were monitored in 16 gerbils, and delayed neuronal death 7 days after the ischemia was studied by light microscopy in these animals. Another group of ten gerbils was subjected to forebrain ischemia to determine the physiologic variables. In experiment 2, microfluorometry was used to investigate the action of lidocaine on a hypoxia-induced intracellular Ca²+ accumulation in gerbil hippocampal slices (ten slices for each).

Animals were anesthetized and maintained with 2% halothane and 98% oxygen. Through a ventral middle cervical incision, both common carotid arteries were exposed and separated carefully from adjacent nerves and tissues. Silk threads (4.0) were looped around them. After the animal was placed in a stereotaxic apparatus (David Kopf Instruments, Tujunga, CA) in the prone position, the skull was exposed and a small burr hole was drilled in the left hemisphere 2 mm anterior and 2 mm lateral to the bregma to insert a thermocouple needle probe (TN-800; Unique Medical Corp., Tokyo, Japan). The tip of the thermocouple needle probe was positioned about 2 mm below the brain surface. An identical probe was inserted into the rectum. Brain and rectal temperatures were carefully maintained at 37–38 [degree sign] Celsius with a heating lamp (Koehler-type illumination lamp; Olympus, Tokyo, Japan). Two additional burr holes were drilled-one in the left hemisphere (0.5 mm posterior and 2 mm lateral to the bregma) to administer drugs and the other in the right hemisphere (2 mm posterior and 2 mm lateral to the bregma) to measure the DC potential shift.

The electrode consisted of a glass micropipette with a tip diameter of about 4 micro meter, which was filled with 3 M KCI with an Ag/AgCl electrode in the barrel. This local electrode was implanted in the right hippocampal CA1 field (2 mm posterior and 2 mm lateral to the bregma, and 2 mm below the brain surface) through the burr hole previously described. The remote electrode (Ag/AgCl) was inserted into the subcutaneous portion of the neck. The DC potential shift was monitored between these electrodes with a model AB-621G DC amplifier (Nihon Kohden, Tokyo, Japan). After a stabilization period of 60 min, animals were assigned to one of two groups. In one, lidocaine (0.8 micro mol in 10 micro liter saline) was administered intracerebroventricularly through a 27-gauge needle inserted into one burr hole. Control animals were given 10 micro liter saline.

Transient forebrain ischemia was achieved by pulling the threads around the bilateral common carotid arteries with 8-g weights. This was done 10 min after lidocaine or saline administration while maintaining the brain and rectal temperatures at 37.5 +/- 0.2 [degree sign] Celsius. [10] After the 4-min period of ischemia, the threads were cut to restore the blood flow. Rectal and brain temperatures were maintained at 37–38 [degree sign] Celsius under halothane anesthesia for 90 min after the reflow. The difference in the DC potential shift was compared by analyzing its onset latency (from the start of ischemia to the sudden depolarization), amplitude, recovery time of depolarization to half-maximal amplitude, and duration at half-maximal amplitude (Figure 1). The thermocouple probes were then gently removed. After all surgical incisions were carefully sutured, the animal was removed from the stereotaxic apparatus. The animal was brought to its cage in a room maintained at constant temperature and allowed access to food and water ad libitum.

Seven days later, the animals were anesthetized with an intraperitoneal injection of sodium pentobarbital. The brains were perfused with heparinized saline and fixed with 10% buffered formalin. After dehydration with graded concentrations of alcohol solutions, the brains were embedded in paraffin. Brain slices, 5 micro meter thick, were stained with hematoxylin and eosin. The number of pyramidal cells in the hippocampal CA1 field per constant area (1 x 1 mm) was counted in healthy animals, and then the number of preserved neurons in the experimental animals was counted in the corresponding area from the coronal section (1.5 mm posterior to bregma). Then the average of percentages of necrotic cells on both sides was obtained for each animal.

Another group of ten gerbils was prepared to determine the physiologic variables that may influence the extent of the neuronal damage in ischemia. The animal was anesthetized with 2% halothane and 98% oxygen. Five animals were given lidocaine intracerebroventricularly, and the remaining five animals were given saline while they were in the prone position using a stereotaxic apparatus. With the animal in the supine position, the animal was prepared for forebrain ischemia. Then, after making an incision in the abdomen, the abdominal aorta was exposed, and a 24-gauge Teflon catheter was inserted into the abdominal aorta to monitor the blood pressure using a model AP-641G blood pressure amplifier (Nihon Kohden, Tokyo, Japan). Ten minutes after the drug was administered, forebrain ischemia for 4 min was induced by clamping the bilateral carotid arteries. Fifteen minutes after reperfusion, the blood sample was collected through the catheter to analyze serum glucose, electrolytes, and arterial blood gas levels according to routine laboratory procedures (blood glucose testing system by electrode, MPG01; Daikin, Osaka, Japan; ABL505 Radiometer; Copenhagen, Denmark). The rectal temperature was maintained at 37–38 [degree sign] Celslius.

Gerbils were anesthetized with ether and then decapitated. The hippocampi were removed rapidly and placed in an ice-cold physiologic medium (124 mM NaCl; 5 mM KCl; 2 mM CaCl²; 2 mM MgCl²; 1.25 mM NaH²PO⁴; 26 mM NaHCO³; 10 mM glucose). Hippocampal transverse slices approximately 300 micro meter thick were cut with a vibrating slicer (DTK-1000; Dosaka Co., Kyoto, Japan). Three to five slices were obtained from each hippocampus and incubated in the physiologic medium equilibrated with a 95% oxygen and 5% carbon dioxide gas mixture for 1 h at 26 [degree sign] Celsius. The slices were preloaded with a fluorescence indicator, rhod-2 acetoxymethyl ester (Dojin, Kumamoto, Japan), which was diluted to 20 micro Meter in the physiologic medium and equilibrated with a 95% oxygen and 5% carbon dioxide gas mixture for 45 min at 26 [degree sign] Celsius. After loading, the slices were further incubated in the physiologic medium for at least 30 min at 26 [degree sign] Celsius.

The [Ca²+]ⁱlevels were measured using an inverted fluorescence microscope, a high-performance video camera, and an image processor system. A low-magnification objective lens (x4) and a side illumination system were used to visualize the fluorescent image of the slice. The slice was transferred to a flow-through chamber (volume [approximately] 0.2 ml) mounted on the fluorescence microscope equipped with a heat plate stage (IMT2; Olympus) and superfused at 3 ml/min with the appropriate medium at 37 [degree sign] Celsius. The temperature of the medium in the chamber was monitored using a thermocouple needle probe (0.4-mm diameter; TN-800; Unique Medical Corp.) and a thermocouple meter (TME-300; Unique Medical Corp.). The slice was excited with 550 nm light produced by an ultraviolet lamp (100 W; Osram, Munich, Germany), filtered by an interference filter (550 nm; band width, < 16 nm) and conducted to the slice through an optic fiber (5-mm diameter). The fluorescence signals (> 580 nm) were captured on a silicon intensified target camera (C2400–8; Hamamatsu Photonics, Hamamatsu, Japan) and processed using an image processor (Argus-100; Hamamatsu Photonics).

Before [Ca²+]ⁱwas measured, the slice loaded with rhod-2 was excited with 550 nm light, and the picture (on a television monitor) was examined to confirm that the dye was uniformly distributed throughout the slice.

Slices were evenly divided into the following four experimental groups. After the slice was placed into the chamber, the slice was perfused with the normoxic medium (a physiologic medium equilibrated with a 95% oxygen and 5% carbon dioxide gas mixture) for 15 min, and the [Ca sup 2+]ⁱin the preischemic state was measured. In vitro hypoxia was induced by switching the normoxic medium to a glucose-free hypoxic medium equilibrated with a 95% nitrogen and 5% carbon dioxide gas mixture. The fluorescence intensity was measured in the image after the induction of in vitro hypoxia. Then the numerical values (pixels) were divided by the value of the corresponding element that had been taken before the measurement. Thus the ratio of [Ca²+]ⁱwas obtained every 10 s.

The effects of lidocaine at concentrations of 10, 50, and 100 micro Meter on the ischemia-like condition were evaluated. After the rhod-2 loading incubation, the slice was perfused with lidocaine-containing normoxic medium for 15 min after placement of the slice into the chamber, and then the medium was switched to the lidocaine-containing ischemia-like medium.

To investigate the effect of lidocaine in a condition free from extracellular Ca²+, the slice was perfused with Ca²+-free medium that was prepared by replacing CaCl²with MgCl²in normoxic and ischemia-like mediums. First, the slice was perfused with the Ca²+-containing normoxic medium for 15 min after placement of the slice in the chamber, and then the medium was changed to the Ca²+-free normoxic medium. After 5 min, the medium was switched to the Ca²+-free ischemia-like medium.

The data obtained from measuring the DC potential were analyzed by unpaired t tests. The data from the histologic experiments were evaluated with the Mann-Whitney test. The data from the fluorometry were analyzed using repeated two-way analysis of variance to detect differences among groups. When differences were found, the Scheffe's test was used post hoc to compare each value with that in the control group.

The forebrain ischemia produced a slow change in the extracellular membrane potential in the early phase. Subsequently, a sudden and marked change was observed in the hippocampal CA1 area. The administration of lidocaine (0.8 micro mol) produced a 140% prolongation of the onset latency of the rapid change in the DC potential shift. The maximal amplitude of the DC potential shift was suppressed to 67% of that in the control group. The recovery time and the duration at half-maximal amplitude of the depolarization revealed no significant differences between the two groups (Figure 1).

Animals in the control group regained consciousness and righting reflex within 30 min after halothane anesthesia was stopped. Similar to the control animals, the animals treated with lidocaine recovered from anesthesia within 30 min. There were no differences between the two groups in taking food and water in the 7 days between ischemia and sacrifice. No seizures were noted in either group. As shown in Table 1, there were no differences in the physiologic variables between the two groups.

Histologically, most of the pyramidal cells had degenerated after 7 days in the control animals subjected to forebrain ischemia. In contrast, the intracerebroventricular administration of lidocaine (0.8 micro mol) 10 min before the ischemia significantly reduced the damage (Figure 2).

When the hippocampal slices were perfused with the in vitro ischemia-like medium, almost no increase in the ratio of [Ca²+]ⁱwas observed in the CA1 field within 240 s after the beginning of the in vitro ischemia. Subsequently, an acute and large increase in [Ca²+] sub i spread throughout the CA1 field, and the ratio reached a plateau (Figure 3and Figure 4). When slices were perfused with the lidocaine-containing medium, the extent of the [Ca²+]ⁱincrease was dose dependently inhibited, with the effects becoming significant at concentrations of 50 and 100 micro Meter. The latencies of the onset of the increase in [Ca²+]ⁱin slices perfused with 50 and 100 micro Meter lidocaine were also prolonged significantly, the values being 392 and 400 s (mean, n = 10), respectively.

When slices were perfused with Ca²+-free in vitro ischemia-like medium, an increase in [Ca²+]ⁱwas observed in the CA1 field similar to that seen in the Ca²+-containing containing condition (Figure 5). The mean value of the latencies of the increase in [Ca²+]ⁱwas 236 s (mean, n = 10). Perfusion with lidocaine (100 micro Meter)-containing medium produced a gradual and moderate increase in [Ca²+]ⁱ. The latency at the beginning of the increase in [Ca²+]ⁱwas markedly prolonged to 384 s (mean, n = 10) by 100 micro Meter lidocaine, and the ratio of fluorescence intensity after 600 s was depressed to 65% of that in the Ca²+-free control.

We have shown the improvement of the ischemia-induced neuronal damage by lidocaine: prolongation of the onset of the AD and suppression of its amplitude. In addition, inhibition of a hypoxia-induced increase in [Ca²+]ⁱby the lidocaine was observed.

The measurement of the DC potential shift between an extracellularly placed electrode in the brain and a remote electrode closely reflects the movement of Na sup +, K sup +, Cl sup -, and Ca²+ across the membrane. [1] In cerebral ischemia, the depletion of adenosine 5'-triphosphate in the central nervous system and glutamate efflux from both neurons and glia progress together [11]; then the sudden depolarization of the neuronal membrane occurs with a huge Ca²+ influx into the neurons. [5] The latter phenomenon causes further adenosine 5'-triphosphate depletion and glutamate efflux. In the present experiment, the intracerebroventricular administration of lidocaine prolonged the onset of the AD and protected the neurons. One previous study showed that the increase in the extracellular concentration of excitatory amino acids such as glutamate and aspartate provoked by ischemia was depressed by the perfusion of this agent through a microdialysis probe. [7] Because these excitatory, neurotransmitters are released on the onset of the AD, the present preischemic administration of lidocaine may contribute to the histologic improvement by delaying cytosolic glutamate release.

In the histologic observation 7 days after the ischemia, transient forebrain ischemia for 4 min at a brain temperature of 37.5 [degree sign] Celsius produced severe damage in the hippocampal CA1 region in control animals. This result corresponds to that in the previous report that transient forebrain ischemia for 3 min in normothermic conditions consistently induced severe damage in the CA1 field in gerbils. [10] The intracerebroventricular administration of lidocaine (0.8 micro mol) 10 min before the ischemia markedly ameliorated this injury. The CA1 area is innervated by glutamatergic fibers, [12] which cause neuronal death in ischemia by their excess release of glutamate, [13] mainly by reversing the Na sup +-cotransport system in a Ca²+-independent manner. [14] Because the blockade of Na sup + channels by tetrodotoxin has been shown to reduce Ca²+-independent excitatory amino acid release, [15] the action of lidocaine by blocking Na sup + channels may take part in the inhibition of the release of excitatory amino acids. The diminution of the amplitude of the AD, which represents the decrease in the ion fluxes, may also be the result of the inhibition of the glutamate release. Lidocaine has been shown to reduce ischemic injury in slices without blocking action potentials, indicating that the amount of lidocaine that needs to improve the damage is less than that required to achieve neural blockade. [16–18] Furthermore, tetrodotoxin suppresses the AD by preventing of the Na sup + load in the early stage of ischemia. [19] These findings indicate that the inhibition of initial events in the ischemic cascade by blocking voltage-dependent Na sup + channels reduces the damage provoked by downstream events.

A sudden and large increase in [Ca²+]ⁱin the CA1 area was observed in the in vitro experiment. This finding corresponds well with the selective vulnerability in this area, because the elevation of [Ca²+]ⁱin postsynaptic neurons after an excess release of glutamate is regarded as a crucial factor in the development of neuronal damage on ischemic attack. [20] Because the administration of lidocaine dose dependently prevented the increase in [Ca²+]ⁱon the extent and the onset, the improvement of the damage by lidocaine in the in vivo experiment may be the result of these inhibitory effects as well as the blockade of Na sup + channels.

The elevation of the cytosolic concentration of Ca²+ in ischemia is induced by both the influx of Ca²+ from the extracellular space and the release of Ca²+ from intracellular stores such as the endoplasmic reticulum and mitochondria. In this study, [Ca²+]ⁱin the Ca²+-free ischemia-like medium increased in a manner similar to that in the Ca²+-containing condition, and the ratio also attained nearly the same value after 600 s. This may indicate that most of the increase in [Ca²+]ⁱis from intracellular sources. However, it is unlikely that the increased amount of cytosolic Ca²+ is provided entirely from the intracellular sources. In the present study, it is not clear that the extracellular concentration of Ca²+ in the microenvironment of the slices decreased by switching the medium to the Ca sup 2+-free medium. In our previous study on recordings of extracellular DC and evoked field potentials combined with measurement of [Ca²+], field excitatory postsynaptic potential (EPSP) disappeared 4 min after switching the normoxic medium to Ca²+-free normoxic medium, indicating that synaptic transmission was depressed by removing extracellular Ca²+. [21] This suggests that the extracellular concentration of Ca²+ is reduced to negligible levels at this point on the electrophysiologic aspect. Although the in vitro hypoxia was induced 5 min after the onset of perfusion with Ca²+-free normoxic medium in the present experiment, [Ca²+]ⁱin the Ca²+-free ischemia-like medium increased in a manner similar to that observed in the Ca²+-containing condition. This similarity may be caused by an insufficient decrease in the extracellular concentration of Ca²+ in the microenvironment of the slices, whereas field EPSP is inhibited at this concentration. The Ca²+ influx is generally presumed to be caused through voltage-gated Ca²+ channels and through agonist-gated Ca²+ channels, mainly N-methyl-D-aspartate-gated Ca²+ channels. Because voltage-gated Ca²+ channels are quickly inactivated during a maintained depolarization, [22] the major component of Ca²+ influx induced by in vitro hypoxia may be through N-methyl-D-aspartate-gated Ca sup 2+ channels.

Two mechanisms of the release of Ca²+ from the endoplasmic reticulum to cytosol have been clarified. [23,24] One is the release through ryanodine receptors, which exist on the membrane of the endoplasmic reticulum. When Ca²+ flows into the cytosol from the extracellular space through mainly N-methyl-D-aspartate-gated Ca²+ channels in an ischemic event, the increase in cytosolic Ca²+ stimulates ryanodine receptors, thereby releasing Ca²+ from intracellular Ca²+ stores (Ca²+-induced Ca²+ release). The other mechanism involves the inositol 1,4,5-triphosphate (IP³) receptors on the endoplasmic reticulum. Stimulation of postsynaptic metabotropic glutamate receptors enhances the activation of phospholipase C, resulting in the facilitation of phosphatidyl-inositol turnover. Thus IP³receptor-linked Ca²+ channels open in response to the increase in the intracellular concentration of IP³, which causes the release of Ca²+(IP³-induced Ca²+ release). However, the mechanism by which lidocaine reduces the release of Ca²+ from the endoplasmic reticulum was not made clear in the present study. There is a report that IP³receptors are highly localized in pyramidal cells in the hippocampal CA1 area and Purkinje cells in the cerebellum, [25] both of which are vulnerable to ischemia. In contrast, ryanodine receptors, which cause Ca²+-induced Ca²+ release, are widely distributed in the whole brain. [26] In another study, lidocaine was shown to facilitate the affinity for ryanodine and the rate of ryanodine association with its binding site in a nonischemic condition. [8] For these reasons, lidocaine seems to inhibit the IP³receptor-mediated Ca²+ release in ischemia.

In this study, lidocaine prevented the increase in [Ca²+] sub i during ischemia, probably as a result of an inhibition of the release of Ca²+ from the intracellular Ca²+ stores and the inhibition of the influx from the extracellular space. The reduction of [Ca²+]ⁱin the early stage of ischemia plays an important role in preventing the cascade leading to neuronal damage, and the present findings contribute to our understanding of the neuro-protective action of lidocaine against ischemia-induced neuronal damage.