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Improvement of ischemic damage in gerbil hippocampal neurons by procaine
Brain Research 792 Ž1998. 16–23
Research report
Improvement of ischemic damage in gerbil hippocampal neurons by procaine Junfeng Chen, Naoto Adachi ) , Keyue Liu, Takumi Nagaro, Tatsuru Arai Department of Anesthesiology and Resuscitology, Ehime UniÕersity School of Medicine, Shitsukawa, Shigenobu-cho, Onsen-gun, Ehime 791-0295, Japan Accepted 23 December 1997
Abstract Acute cerebral ischemia induces membrane depolarization in the neuron, thereby incurring the simultaneous influx of various ions such as Naq and Ca2q. Since procaine possesses the ability to inhibit the release of Ca2q from intracellular Ca2q stores to the cytosol as well as the ability to block Naq channels, the effects of procaine on ischemia were investigated in the present study in gerbils both in vivo and in vitro. The histologic outcome was evaluated 7 days after 3 min of transient forebrain ischemia by assessing delayed neuronal death in hippocampal CA1 pyramidal cells in animals administered procaine Ž0.2, 0.4, or 2 m mol. intracerebroventricularly 10 min before ischemia and in animals given saline. The changes in the direct-current potential shift in the hippocampal CA1 area were measured using an identical animal model. A hypoxia-induced intracellular Ca2q increase was evaluated by in vitro microfluorometry in gerbil hippocampal slices, and the effects of procaine Ž10, 50, and 100 m molrl. on the Ca2q accumulation were examined. Additionally, the effect of procaine Ž100 m molrl. in a Ca2q-free condition was investigated. The histologic outcome was improved and the onset of the ischemia-induced membrane depolarization was prolonged by the preischemic administration of procaine. The increase in the intracellular concentration of Ca2q induced by the in vitro hypoxia was suppressed by the perfusion of procaine-containing mediums Ž50 and 100 m molrl., regarding both the initiation and the extent of the increase. A hypoxia-induced intracellular Ca2q elevation in the Ca2q-free condition was observed, and the perfusion with procaine Ž100 m molrl. inhibited this elevation. Procaine helps protect neurons from ischemia by suppressing the direct-current potential shift and by inhibiting the release of Ca2q from the intracellular Ca2q stores, as well as by inhibiting the influx of Ca2q from the extracellular space. q 1998 Elsevier Science B.V. Keywords: Anoxic depolarization; Ca2q; Cerebral ischemia; Gerbil; Hippocampus; Procaine
1. Introduction The homeostasis of intracellular and extracellular ions is essential for the maintenance of neuronal functions, which is supported by adequate supplies of oxygen and glucose. In cerebral ischemia, energy failure triggers depolarization of the neuronal membrane because of the inadequate active transportation of ions and influxes of ions such as Naq and Ca2q w7,23x. Simultaneously, various excitatory neurotransmitters are released w1,2,19x, which causes a marked influx of Ca2q into postsynaptic neurons. These events provoke the catastrophic enzymatic process leading to irreversible neuronal damage w17x. Procaine has been used as an agent for analgesia, intravenous anesthesia, and antiarrhythmia w3,28x. Local anesthetics possess the ability to block Naq channels, which is regarded as a mechanism underlying the prevention of ischemic neuronal damage w6,11,12x. The increase
in the intracellular concentration of Ca2q ŽwCa2q x i ., which is caused by the release of Ca2q from intracellular stores as well as the influx of Ca2q from the extracellular space, plays an important role in the development of neuronal injury w16x. However, there are some differences among local anesthetics in their effects on the release of Ca2q from intracellular stores. Procaine inhibits ryanodine binding to its receptors, which causes the release of Ca2q from the endoplasmic reticulum, whereas lidocaine enhances ryanodine binding to its receptor w21x. The purpose of the present study was to characterize the in vivo effects of procaine on the ischemia-induced histologic outcome and alterations of the direct-current ŽDC. potential shift Žanoxic depolarization; AD., and to investigate the in vitro effects of procaine on a hypoxia-induced increase in the wCa2q x i . 2. Materials and methods 2.1. Animals
X
Abbreviations: AD, anoxic depolarization; ATP, adenosine 5 -triphosphate; DC, direct current; IP3 , inositol 1,4,5-triphosphate ) Corresponding author. Fax: q81-89-960-5386. 0006-8993r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved. PII S 0 0 0 6 - 8 9 9 3 Ž 9 8 . 0 0 0 1 1 - 0
This study was approved by the Committee on Animal Experimentation at Ehime University School of Medicine,
J. Chen et al.r Brain Research 792 (1998) 16–23
Ehime, Japan. Male Mongolian gerbils weighing 60–80 g ŽSeiwa Experimental Animals, Fukuoka, Japan. were housed in groups in a room controlled at 23 " 18C with a 12-h lightr12-h dark cycle Žlights on at 6:00 h.. The animals were deprived of food at least 6 h before the ischemia, because of the influence of hyperglycemia on ischemic brain damage w18,22x. All in vivo experiments were performed under spontaneous ventilation. In experiment 1, delayed neuronal death in the hippocampal CA1 sector provoked by forebrain ischemia was examined by light microscopy. In experiment 2, the changes in the DC potential produced by forebrain ischemia in the hippocampal CA1 region were monitored. In experiment 3, microfluorometry was used to investigate the action of procaine on a hypoxia-induced intracellular Ca2q accumulation in gerbil hippocampal slices. 2.2. Experiment 1: In ÕiÕo experiment on histologic outcome
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animal was removed from the stereotaxic apparatus and the surgical incisions were sutured carefully. The animal was then brought to its cage in a room maintained at constant temperature and allowed access to food and water ad libitum. Seven days after the transient forebrain ischemia, 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 brain was embedded in paraffin. Brain slices, 5 m m thick, were stained with hematoxylin and eosin. The numbers of preserved pyramidal cells in the hippocampal CA1 field per 1 mm length of stratum pyramidale in each hemisphere were counted in the same level of coronal section Ž1.5 mm posterior to the bregma. in a single blinded manner. The average of values on both sides was then obtained for each animal. 2.3. Experiment 2: Measurement of the DC potential
In this experiment, 40 gerbils were prepared and then assigned to 4 groups. The 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 Ž4.0. were looped around these arteries. After the animal was placed in a stereotaxic apparatus ŽDavid Kopf Instruments, Tujunga, CA, USA. in the prone position, the skull was exposed and a small burr hole was drilled in the right hemisphere at 2 mm anterior and 2 mm lateral to the bregma for the insertion of a thermocouple needle probe ŽTN-800; Unique Medical, 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. The brain and rectal temperatures were carefully maintained at 37.5 " 0.28C with a heating lamp ŽKoehler type illumination lamp; Olympus, Tokyo, Japan.. Another burr bole was drilled in the left hemisphere Ž0.5 mm posterior and 2.5 mm lateral to the bregma. for drug administration. After a stabilization period of 30 min, procaine was administered intracerebroventricularly through the burr hole at a depth of 2.5 mm in a constant volume of 10 m l via a 27-gauge needle. The rate of the injection was 5 m lrmin. The doses of procaine were 0.2 m mol Ž10 gerbils., 0.4 m mol Ž10 gerbils. and 2 m mol Ž10 gerbils.. Control animals Ž10 gerbils. received 10 m l of saline. Transient forebrain ischemia Žfor a 3-min period. was achieved by pulling the threads around the bilateral common carotid arteries with 8-g weights 10 min after the drug administration, while maintaining the brain and rectal temperatures at 37.5 " 0.28C. After the 3-min ischemia, the threads were cut to restore the blood flow. The brain and rectal temperatures were maintained at 37.5 " 0.28C under halothane anesthesia for 30 min after the reflow. The thermocouple probes were then gently pulled out. The
Forty gerbils were subjected to the measurement of the DC potential in the hippocampal CA1 area. After being anesthetized, the animals were prepared for forebrain ischemia by the same procedure as that described in experiment 1. After the animal was fixed in the stereotaxic apparatus, a small burr hole was drilled in the right hemisphere at 2 mm posterior and 2 mm lateral to the bregma. The electrode consisted of a glass micropipette with a tip diameter of about 4 m m, which was filled with 2 molrl NaCl with an AgrAgCl electrode in the barrel. This local electrode was inserted through the drilled hole and was placed 2 mm below the brain surface. The remote electrode ŽAgrAgCl. was inserted subcutaneously at the back of the neck. After a stabilization period for 30 min, procaine Ž0.2, 0.4, or 2 m mol. was administered intracerebroventricularly in a volume of 10 m l, and the control animals were given saline. Transient forebrain ischemia for 3 min was performed 10 min after the injection, while the brain and rectal temperatures were maintained at 37.5 " 0.28C. The DC potential was recorded with a modal AB621G DC amplifier ŽNihon Kohden, Tokyo, Japan.. The difference in the DC potential shift was compared regarding its onset latency, amplitude, recovery time of the depolarization to the half-maximal amplitude, and the duration of the half-maximal amplitude. 2.4. Experiment 3: Measurement of the [Ca 2 q ]i Gerbils were anesthetized with ether and decapitated. The brain was rapidly removed and placed in an ice-cold physiological medium Žmmolrl: NaCl 124; KCl 5; CaCl 2 2; MgCl 2 2; NaH 2 PO4 1.25; NaHCO 3 26; glucose 10.. Hippocampal transverse slices, approximately 300 m m thick, were cut with a vibrating slicer ŽDTK-1000; Dosaka, Kyoto, Japan.; 3–5 slices were obtained from each hip-
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J. Chen et al.r Brain Research 792 (1998) 16–23
pocampus. The slices were incubated in the physiological medium equilibrated with a 95% O 2r5% CO 2 gas mixture for 1 h at 268C. The slices were preloaded with a fluores-
Fig. 2. Effects of procaine Ž0.2, 0.4, and 2.0 m mol., intracerebroventricularly administered 10 min before ischemia on the delayed neuronal death of CA1 pyramidal cells. CA1 pyramidal cells were examined 7 days after the 3-min ischemia, and the number of pyramidal cells Žordinate. was determined. Values obtained from individual animals are shown. The number of pyramidal cells in the CA1 area in intact animals was 256"12 Žmean"S.D., ns 5.. Cont, saline-injected control group. ) p- 0.05, )) p- 0.01 compared with the control group.
Fig. 1. Typical photographs showing the effects of procaine. ŽA. Salineinjected control animal, ŽB. 0.2 m mol, ŽC. 0.4 m mol, ŽD. 2.0 m mol of procaine. Bar s 0.1 mm.
cent indicator, rhod-2 acetoxymethyl ester ŽDojin, Kumamoto, Japan. which was diluted to 20 m molrl in the physiological medium and equilibrated with a 95% O 2r5% CO 2 gas mixture for 45 min at 268C. Following loading, the slices were further incubated in the physiological medium for at least 30 min at 268C. The wCa2q x i levels were measured using an inverted fluorescence microscope, a high-performance video camera and an image processor system. A low-magnification objective lens Ž=4. and a side illumination system were used to visualize the fluorescence image of the slice. The slice was transferred to a flow-through chamber Žvolume ; 0.2 ml. mounted on the fluorescence microscope equipped with a heat plate stage ŽIMT2; Olympus. and superfused at 3 mlrmin with the appropriate medium at 36.58C. The temperature of the medium in the chamber was monitored using a thermocouple needle probe. The slice was excited with 550 nm light produced by an ultraviolet ŽUV. lamp Ž100 W; Osram, Munich, Germany., filtered by an interference filter Ž550 nm, band width - 16 nm., and conducted to the slice through an optic fiber Ž5 mm diameter.. The fluorescence signals Ž) 580 nm. were captured on an SIT Žsilicon-intensified target. camera ŽC2400-8; Hamamatsu Photonics, Hamamatsu, Japan., and processed using an image processor ŽArgus-100; Hamamatsu Photonics.. Prior to the measurement of the wCa2q x i , the slice loaded with rhod-2 was excited with 550 nm light, and the picture Žon a TV monitor. was examined to confirm that the dye was uniformly distributed throughout the slice. After placement of the slice into the chamber, the slice was perfused with the normoxic medium Ža physiological medium equilibrated with a 95% O 2r5% CO 2 gas mixture. for 15 min, and the wCa2q x i in the preischemic state was measured. In vitro hypoxia was induced by switching
J. Chen et al.r Brain Research 792 (1998) 16–23
the normoxic medium to a glucose-free hypoxic medium equilibrated with a 95% N2r5% CO 2 gas mixture. The fluorescence intensity was measured in the image after the induction of in vitro hypoxia. Then, the numerical values Žpixels. were divided by the value of the corresponding element that had been taken prior to the measurement. Thus, the ratio of the wCa2q x i was obtained every 10 s. The effects of procaine at concentrations of 10, 50, and 100 m molrl on the ischemia-like condition were evaluated. Following the rhod-2 loading incubation, the slice was perfused with procaine-containing normoxic medium for 15 min after placement of the slice into the chamber, and then the medium was switched to the procaine-containing ischemia-like medium. To investigate the effect of procaine in a condition free from extracellular Ca2q, the slice was perfused with Ca2q-free mediums that were prepared by replacing the CaCl 2 with MgCl 2 in both normoxic and ischemia-like mediums. First, the slice was perfused with the Ca2q-containing normoxic medium for 15 min after placement of the slice in the chamber, and then the medium was changed to the Ca2q-free normoxic medium. After 5 min, the medium was switched to the Ca2q-free ischemia-like medium. The effect of procaine was evaluated by adding procaine to each medium. 2.5. Statistical analysis The data obtained from the histology were evaluated with the Kruskal–Wallis test followed by the Mann–Whitney test. The data obtained by measuring the DC potential were analyzed by analysis of variance followed by Dunnett’s test. The data from the fluorometry were analyzed using a repeated two-way analysis of variance to detect differences among groups. When differences were found, Scheffe’s ´ test was used post hoc to compare each value with that in the control group.
3. Results Figs. 1 and 2 show the extent of neuronal damage in the hippocampal CA1 region in each group. In the control animals, almost all pyramidal cells were degenerated 7
Fig. 3. The typical change in the DC potential shift produced by 3 min of transient forebrain ischemia.
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Table 1 Effects of procaine on the anoxic depolarization Onset Žs. Control 53"16 Procaine 0.2 m mol 71"13 0.4 m mol 79"14) 2.0 m mol 102"27))
Amplitude ŽmV. Recovery Žs. Duration Žs. 22"11
101"39
206"53
19"10 19"7 22"10
94"32 117"51 116"44
176"37 193"61 164"50
The differences in the DC potential shift were compared regarding its onset latency, amplitude, recovery time of the depolarization to half-maximal amplitude, and the duration of the half-maximal amplitude. ) p0.05, )) p- 0.01 compared with the respective values in the control group.
days after ischemia. The number of preserved neurons was 8 " 6 Žmean " S.D., n s 10.. In contrast, the varying doses of procaine reduced the damage significantly. The numbers of preserved neurons were 53 " 23 Žprocaine 0.2 m mol., 181 " 35 Žprocaine 0.4 m mol., and 246 " 14 Žprocaine 2 m mol., and there were no differences between the both sides. In the animals that received 2 m mol of procaine, almost all pyramidal cells remained intact 7 days after ischemia. However, in 8 of these 10 gerbils, the respiration stopped for about 1 min after the intracerebroventricular administration of procaine Ž2 m mol., and the animals started breathing spontaneously after about 1 min. As shown in Fig. 3, the forebrain ischemia provoked a gradual decrease in the extracellular membrane potential in the hippocampal CA1 area. Then, the DC potential shifted suddenly. After reperfusion, the membrane repolarized gradually. In the animals that received procaine Ž0.2, 0.4, or 2 m mol., the onset latencies were prolonged by 37%, 49%, and 100%, respectively, compared with the control group ŽTable 1.. However, the maximal amplitude of the DC potential shift, the recovery time of the depolarization, and the duration of the half-maximal amplitude were not significantly different among all groups. Fig. 4 is comprised of photographs showing the elevation of the wCa2q x i in hippocampal slices induced by the in vitro ischemia-like condition. As shown in Fig. 5, when the hippocampal slice was perfused with the in vitro ischemia-like medium, almost no increase in the ratio of the wCa2q x i was observed in the CA1 field within 250 s after the beginning of the in vitro hypoxia. Subsequently, an acute and large increase in the wCa2q x i spread throughout the CA1 field, and the ratio reached a plateau. When slices were perfused with the procaine-containing medium, the onset and the extent of the wCa2 x i increase were dose-dependently inhibited with the effect being significant at the procaine concentrations of 50 and 100 m molrl. As shown in Fig. 6, when the slices were perfused with Ca2q-free in vitro ischemia-like medium, an increase in the wCa2q x i was observed in the CA1 field in a manner similar to that in the Ca2q-containing condition. Perfusion with procaine Ž100 m molrl.-containing medium produced a
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J. Chen et al.r Brain Research 792 (1998) 16–23
Fig. 4. Photographs showing the difference between a control slice and a procaine Ž100 m molrl.-perfusing slice in the wCa2q x i elevation induced by in vitro hypoxia. ŽA. 250 s, ŽB. 300 s, ŽC. 350 s, ŽD. 400 s, and ŽE. 450 s after the start of hypoxia. The open rectangles represent the measured areas. Bar s 0.5 mm.
J. Chen et al.r Brain Research 792 (1998) 16–23
Fig. 5. Changes in the ratio of the wCa2q x i in slices of the gerbil hippocampal CA1 field which underwent in vitro ischemia-like conditions. The standard ischemia-like medium Ž`., procaine Ž10 m molrl.containing ischemia-like medium Žv ., procaine Ž50 m molrl.-containing ischemia-like medium Ž'., and procaine Ž100 m molrl.-containing ischemia-like medium ŽB. effects are shown. Each value represents the mean"S.D. of 10 slices. ) p- 0.05, )) p- 0.01, ))) p- 0.001 compared with the respective values in the standard ischemia-like group.
Fig. 6. Changes in the ratio of wCa2q x i in slices of the hippocampal CA1 field which underwent Ca2q-free in vitro ischemia-like conditions. The Ca2q-free in vitro ischemia-like medium Ž`., and procaine Ž100 m molrl.-containing Ca2q-free in vitro ischemia-like medium Žv . effects are shown. Each value represents the mean"S.D. of 8 or 10 slices. ) p- 0.05, )) p- 0.01, ))) p- 0.001 compared with the respective values in the Ca2q-free ischemia-like group.
gradual and moderate increase in the wCa2q x i . The latency at the beginning of the increase in the wCa2q x i was markedly prolonged, and the extent of the increase was also depressed by 100 m molrl of procaine.
4. Discussion In the present study, we observed the improvement of the ischemia-induced damage in hippocampal CA1 pyramidal cells and a prolongation of the onset of the AD in the in vivo experiments. We also observed the inhibition of the hypoxia-induced increase in the wCa2q x i by procaine in the in vitro experiments. The brain temperature was carefully maintained at 37.58C in all experiments, because the brain temperature in ischemia and reperfusion plays an important role in the histologic outcome w15x. Under this normothermic condi-
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tion, transient forebrain ischemia for 3 min produced a marked damage in the hippocampal CA1 region in control animals, which is consistent with a previous report w15x. In contrast, the intracerebroventricular administration of procaine dose-dependently ameliorated the neuronal damage, although a high dose of procaine Ž2 m mol. inhibited the spontaneous respiration in animals. The hippocampal CA1 area is vulnerable to ischemia, and this region is innervated by glutamatergic fibers w24x, which cause neuronal death by their excess release of glutamate w9x. The excess release of glutamate in ischemia has been shown to be caused mainly by the reversal of the Naq-cotransport system in a Ca2q-independent manner w20x, and the blockade of Naq channels by tetrodotoxin or lidocaine reduces the Ca2q-independent excitatory amino acid release w5,13,25,27x. Therefore, the blockade of Naq channels by procaine may take part in the inhibition of the release of excitatory amino acid, thereby reducing the glutamate toxicity. The Naq gradient across the cell membrane is an energy source for Naq–Ca2q and Naq–Kq exchange as well as glutamate uptake from the extracellular space. The Naq–Kq ATPase pump uses ATP to maintain the Naq gradient. In the early period of ischemia, the function of the ion pump is maintained by the cellular storage of ATP. When ATP is depleted, the ion pump loses its function and various neurotransmitters are released, which cause a sudden depolarization of the membrane w5,7,17,23x. In the present experiment, we found that the intracerebroventricular administration of procaine prolonged the onset latency of the sudden shift of the DC potential. The protective effect of this agent may be due to the prolongation of the onset of the AD. The inhibition of the Naq influx through Naq channels by procaine may block the initiation of the catastrophic enzymatic process leading to neuronal damage. It has been generally believed that an excessive increase in the wCa2q x i leads to irreversible neuronal injury w4,10x. In our present in vitro experiment, we found a sudden and large increase in the wCa2q x i in the CA1 area in the ischemia-like condition. This finding is in good agreement with the selective vulnerability in this area. The extracellular DC potential shift closely reflects the movement of Naq, Kq, Cly and Ca2q across the membrane w7x, and the acute and large negative shift of the membrane potential has been shown to be accompanied by an acute elevation of the wCa2q x i w17x. Therefore, the sudden shift of the AD observed in the in vivo experiment is closely related to the sudden increase in the wCa2q x i in the in vitro experiment. For these reasons, the protective effects of procaine in the in vivo experiment are thought to have been caused by the prevention of the increase in the wCa2q x i . It has been demonstrated that a large elevation of the wCa2q x i induced by cerebral ischemia in the CA1 field of the hippocampus was caused by both the Ca2q influx from the extracellular space and the Ca2q release from the
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J. Chen et al.r Brain Research 792 (1998) 16–23
intracellular Ca2q stores such as the endoplasmic reticulum and mitochondria w16x. A similar phenomenon was observed in the present study. The wCa2q x i increased in the Ca2q-free hypoxic condition as well as in the Ca2q-containing condition, although the extent of the increase was smaller than that in the Ca2q-containing condition. Therefore, it is speculated that the Ca2q release from intracellular stores plays an important role in the elevation of the wCa2q x i in ischemia, as does the influx of Ca2q from the extracellular space. There are two mechanisms, by which Ca2q is released from the endoplasmic reticulum to the cytosol w8,26x. One is the release through ryanodine receptors, which exist on the membrane of the endoplasmic reticulum. The increase in the cytosolic concentration of Ca2q, which is caused by the influx of Ca2q from the extracellular space through mainly N-methyl-D-aspartate ŽNMDA.-gated Ca2q channels in an ischemic event, stimulates the release of Ca2q from the endoplasmic reticulum by activating ryanodine receptors ŽCa2q-induced Ca2q release.. The other mechanism involves the inositol 1,4,5-triphosphate ŽIP3 . receptors on the endoplasmic reticulum. The stimulation of postsynaptic metabotropic glutamate receptors activates the intracellular phosphatidylinositol ŽPI. turnover. Thus, IP3 receptor-linked Ca2q channels open in response to the increase in the intracellular concentration of IP3 , resulting in the release of Ca2q ŽIP3-induced Ca2q release.. The mechanism by which procaine reduces the release of Ca2q from the intracellular store was not elucidated in the present study. However, procaine has been shown to be a blocker of ryanodine receptors, and to inhibit ryanodinemediated Ca2q release from the endoplasmic reticulum. Procaine has also been demonstrated not to affect IP3mediated Ca2q release w14,21,29x. Therefore, procaine may inhibit the ryanodine-mediated Ca2q release in ischemia. In this study, the protective effects of procaine on hippocampal CA1 pyramidal cells against ischemia by inhibiting the onset time of the AD and preventing the increase in the wCa2q x i were observed. The reduction of the wCa2q x i in the early stage of ischemia seems to play an important role in preventing the cascade leading to neuronal damage, and the present findings contribute to the clarification of the neuroprotective action of procaine against ischemia-induced neuronal damage.
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and phenytoin without blockade of action potentials, Brain Res. 664 Ž1994. 167–177. w28x M.G. Wright, Clinical use of procaine, Br. J. Hosp. Med. 53 Ž1995. 296. w29x C. Yue, K.L. White, W.A. Reed, T.D. Bunch, The existence of inositol 1,4,5-triphosphate and ryanodine receptors in mature bovine oocytes, Development 121 Ž1995. 2645–2654.
Research report
Improvement of ischemic damage in gerbil hippocampal neurons by procaine Junfeng Chen, Naoto Adachi ) , Keyue Liu, Takumi Nagaro, Tatsuru Arai Department of Anesthesiology and Resuscitology, Ehime UniÕersity School of Medicine, Shitsukawa, Shigenobu-cho, Onsen-gun, Ehime 791-0295, Japan Accepted 23 December 1997
Abstract Acute cerebral ischemia induces membrane depolarization in the neuron, thereby incurring the simultaneous influx of various ions such as Naq and Ca2q. Since procaine possesses the ability to inhibit the release of Ca2q from intracellular Ca2q stores to the cytosol as well as the ability to block Naq channels, the effects of procaine on ischemia were investigated in the present study in gerbils both in vivo and in vitro. The histologic outcome was evaluated 7 days after 3 min of transient forebrain ischemia by assessing delayed neuronal death in hippocampal CA1 pyramidal cells in animals administered procaine Ž0.2, 0.4, or 2 m mol. intracerebroventricularly 10 min before ischemia and in animals given saline. The changes in the direct-current potential shift in the hippocampal CA1 area were measured using an identical animal model. A hypoxia-induced intracellular Ca2q increase was evaluated by in vitro microfluorometry in gerbil hippocampal slices, and the effects of procaine Ž10, 50, and 100 m molrl. on the Ca2q accumulation were examined. Additionally, the effect of procaine Ž100 m molrl. in a Ca2q-free condition was investigated. The histologic outcome was improved and the onset of the ischemia-induced membrane depolarization was prolonged by the preischemic administration of procaine. The increase in the intracellular concentration of Ca2q induced by the in vitro hypoxia was suppressed by the perfusion of procaine-containing mediums Ž50 and 100 m molrl., regarding both the initiation and the extent of the increase. A hypoxia-induced intracellular Ca2q elevation in the Ca2q-free condition was observed, and the perfusion with procaine Ž100 m molrl. inhibited this elevation. Procaine helps protect neurons from ischemia by suppressing the direct-current potential shift and by inhibiting the release of Ca2q from the intracellular Ca2q stores, as well as by inhibiting the influx of Ca2q from the extracellular space. q 1998 Elsevier Science B.V. Keywords: Anoxic depolarization; Ca2q; Cerebral ischemia; Gerbil; Hippocampus; Procaine
1. Introduction The homeostasis of intracellular and extracellular ions is essential for the maintenance of neuronal functions, which is supported by adequate supplies of oxygen and glucose. In cerebral ischemia, energy failure triggers depolarization of the neuronal membrane because of the inadequate active transportation of ions and influxes of ions such as Naq and Ca2q w7,23x. Simultaneously, various excitatory neurotransmitters are released w1,2,19x, which causes a marked influx of Ca2q into postsynaptic neurons. These events provoke the catastrophic enzymatic process leading to irreversible neuronal damage w17x. Procaine has been used as an agent for analgesia, intravenous anesthesia, and antiarrhythmia w3,28x. Local anesthetics possess the ability to block Naq channels, which is regarded as a mechanism underlying the prevention of ischemic neuronal damage w6,11,12x. The increase
in the intracellular concentration of Ca2q ŽwCa2q x i ., which is caused by the release of Ca2q from intracellular stores as well as the influx of Ca2q from the extracellular space, plays an important role in the development of neuronal injury w16x. However, there are some differences among local anesthetics in their effects on the release of Ca2q from intracellular stores. Procaine inhibits ryanodine binding to its receptors, which causes the release of Ca2q from the endoplasmic reticulum, whereas lidocaine enhances ryanodine binding to its receptor w21x. The purpose of the present study was to characterize the in vivo effects of procaine on the ischemia-induced histologic outcome and alterations of the direct-current ŽDC. potential shift Žanoxic depolarization; AD., and to investigate the in vitro effects of procaine on a hypoxia-induced increase in the wCa2q x i . 2. Materials and methods 2.1. Animals
X
Abbreviations: AD, anoxic depolarization; ATP, adenosine 5 -triphosphate; DC, direct current; IP3 , inositol 1,4,5-triphosphate ) Corresponding author. Fax: q81-89-960-5386. 0006-8993r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved. PII S 0 0 0 6 - 8 9 9 3 Ž 9 8 . 0 0 0 1 1 - 0
This study was approved by the Committee on Animal Experimentation at Ehime University School of Medicine,
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Ehime, Japan. Male Mongolian gerbils weighing 60–80 g ŽSeiwa Experimental Animals, Fukuoka, Japan. were housed in groups in a room controlled at 23 " 18C with a 12-h lightr12-h dark cycle Žlights on at 6:00 h.. The animals were deprived of food at least 6 h before the ischemia, because of the influence of hyperglycemia on ischemic brain damage w18,22x. All in vivo experiments were performed under spontaneous ventilation. In experiment 1, delayed neuronal death in the hippocampal CA1 sector provoked by forebrain ischemia was examined by light microscopy. In experiment 2, the changes in the DC potential produced by forebrain ischemia in the hippocampal CA1 region were monitored. In experiment 3, microfluorometry was used to investigate the action of procaine on a hypoxia-induced intracellular Ca2q accumulation in gerbil hippocampal slices. 2.2. Experiment 1: In ÕiÕo experiment on histologic outcome
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animal was removed from the stereotaxic apparatus and the surgical incisions were sutured carefully. The animal was then brought to its cage in a room maintained at constant temperature and allowed access to food and water ad libitum. Seven days after the transient forebrain ischemia, 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 brain was embedded in paraffin. Brain slices, 5 m m thick, were stained with hematoxylin and eosin. The numbers of preserved pyramidal cells in the hippocampal CA1 field per 1 mm length of stratum pyramidale in each hemisphere were counted in the same level of coronal section Ž1.5 mm posterior to the bregma. in a single blinded manner. The average of values on both sides was then obtained for each animal. 2.3. Experiment 2: Measurement of the DC potential
In this experiment, 40 gerbils were prepared and then assigned to 4 groups. The 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 Ž4.0. were looped around these arteries. After the animal was placed in a stereotaxic apparatus ŽDavid Kopf Instruments, Tujunga, CA, USA. in the prone position, the skull was exposed and a small burr hole was drilled in the right hemisphere at 2 mm anterior and 2 mm lateral to the bregma for the insertion of a thermocouple needle probe ŽTN-800; Unique Medical, 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. The brain and rectal temperatures were carefully maintained at 37.5 " 0.28C with a heating lamp ŽKoehler type illumination lamp; Olympus, Tokyo, Japan.. Another burr bole was drilled in the left hemisphere Ž0.5 mm posterior and 2.5 mm lateral to the bregma. for drug administration. After a stabilization period of 30 min, procaine was administered intracerebroventricularly through the burr hole at a depth of 2.5 mm in a constant volume of 10 m l via a 27-gauge needle. The rate of the injection was 5 m lrmin. The doses of procaine were 0.2 m mol Ž10 gerbils., 0.4 m mol Ž10 gerbils. and 2 m mol Ž10 gerbils.. Control animals Ž10 gerbils. received 10 m l of saline. Transient forebrain ischemia Žfor a 3-min period. was achieved by pulling the threads around the bilateral common carotid arteries with 8-g weights 10 min after the drug administration, while maintaining the brain and rectal temperatures at 37.5 " 0.28C. After the 3-min ischemia, the threads were cut to restore the blood flow. The brain and rectal temperatures were maintained at 37.5 " 0.28C under halothane anesthesia for 30 min after the reflow. The thermocouple probes were then gently pulled out. The
Forty gerbils were subjected to the measurement of the DC potential in the hippocampal CA1 area. After being anesthetized, the animals were prepared for forebrain ischemia by the same procedure as that described in experiment 1. After the animal was fixed in the stereotaxic apparatus, a small burr hole was drilled in the right hemisphere at 2 mm posterior and 2 mm lateral to the bregma. The electrode consisted of a glass micropipette with a tip diameter of about 4 m m, which was filled with 2 molrl NaCl with an AgrAgCl electrode in the barrel. This local electrode was inserted through the drilled hole and was placed 2 mm below the brain surface. The remote electrode ŽAgrAgCl. was inserted subcutaneously at the back of the neck. After a stabilization period for 30 min, procaine Ž0.2, 0.4, or 2 m mol. was administered intracerebroventricularly in a volume of 10 m l, and the control animals were given saline. Transient forebrain ischemia for 3 min was performed 10 min after the injection, while the brain and rectal temperatures were maintained at 37.5 " 0.28C. The DC potential was recorded with a modal AB621G DC amplifier ŽNihon Kohden, Tokyo, Japan.. The difference in the DC potential shift was compared regarding its onset latency, amplitude, recovery time of the depolarization to the half-maximal amplitude, and the duration of the half-maximal amplitude. 2.4. Experiment 3: Measurement of the [Ca 2 q ]i Gerbils were anesthetized with ether and decapitated. The brain was rapidly removed and placed in an ice-cold physiological medium Žmmolrl: NaCl 124; KCl 5; CaCl 2 2; MgCl 2 2; NaH 2 PO4 1.25; NaHCO 3 26; glucose 10.. Hippocampal transverse slices, approximately 300 m m thick, were cut with a vibrating slicer ŽDTK-1000; Dosaka, Kyoto, Japan.; 3–5 slices were obtained from each hip-
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pocampus. The slices were incubated in the physiological medium equilibrated with a 95% O 2r5% CO 2 gas mixture for 1 h at 268C. The slices were preloaded with a fluores-
Fig. 2. Effects of procaine Ž0.2, 0.4, and 2.0 m mol., intracerebroventricularly administered 10 min before ischemia on the delayed neuronal death of CA1 pyramidal cells. CA1 pyramidal cells were examined 7 days after the 3-min ischemia, and the number of pyramidal cells Žordinate. was determined. Values obtained from individual animals are shown. The number of pyramidal cells in the CA1 area in intact animals was 256"12 Žmean"S.D., ns 5.. Cont, saline-injected control group. ) p- 0.05, )) p- 0.01 compared with the control group.
Fig. 1. Typical photographs showing the effects of procaine. ŽA. Salineinjected control animal, ŽB. 0.2 m mol, ŽC. 0.4 m mol, ŽD. 2.0 m mol of procaine. Bar s 0.1 mm.
cent indicator, rhod-2 acetoxymethyl ester ŽDojin, Kumamoto, Japan. which was diluted to 20 m molrl in the physiological medium and equilibrated with a 95% O 2r5% CO 2 gas mixture for 45 min at 268C. Following loading, the slices were further incubated in the physiological medium for at least 30 min at 268C. The wCa2q x i levels were measured using an inverted fluorescence microscope, a high-performance video camera and an image processor system. A low-magnification objective lens Ž=4. and a side illumination system were used to visualize the fluorescence image of the slice. The slice was transferred to a flow-through chamber Žvolume ; 0.2 ml. mounted on the fluorescence microscope equipped with a heat plate stage ŽIMT2; Olympus. and superfused at 3 mlrmin with the appropriate medium at 36.58C. The temperature of the medium in the chamber was monitored using a thermocouple needle probe. The slice was excited with 550 nm light produced by an ultraviolet ŽUV. lamp Ž100 W; Osram, Munich, Germany., filtered by an interference filter Ž550 nm, band width - 16 nm., and conducted to the slice through an optic fiber Ž5 mm diameter.. The fluorescence signals Ž) 580 nm. were captured on an SIT Žsilicon-intensified target. camera ŽC2400-8; Hamamatsu Photonics, Hamamatsu, Japan., and processed using an image processor ŽArgus-100; Hamamatsu Photonics.. Prior to the measurement of the wCa2q x i , the slice loaded with rhod-2 was excited with 550 nm light, and the picture Žon a TV monitor. was examined to confirm that the dye was uniformly distributed throughout the slice. After placement of the slice into the chamber, the slice was perfused with the normoxic medium Ža physiological medium equilibrated with a 95% O 2r5% CO 2 gas mixture. for 15 min, and the wCa2q x i in the preischemic state was measured. In vitro hypoxia was induced by switching
J. Chen et al.r Brain Research 792 (1998) 16–23
the normoxic medium to a glucose-free hypoxic medium equilibrated with a 95% N2r5% CO 2 gas mixture. The fluorescence intensity was measured in the image after the induction of in vitro hypoxia. Then, the numerical values Žpixels. were divided by the value of the corresponding element that had been taken prior to the measurement. Thus, the ratio of the wCa2q x i was obtained every 10 s. The effects of procaine at concentrations of 10, 50, and 100 m molrl on the ischemia-like condition were evaluated. Following the rhod-2 loading incubation, the slice was perfused with procaine-containing normoxic medium for 15 min after placement of the slice into the chamber, and then the medium was switched to the procaine-containing ischemia-like medium. To investigate the effect of procaine in a condition free from extracellular Ca2q, the slice was perfused with Ca2q-free mediums that were prepared by replacing the CaCl 2 with MgCl 2 in both normoxic and ischemia-like mediums. First, the slice was perfused with the Ca2q-containing normoxic medium for 15 min after placement of the slice in the chamber, and then the medium was changed to the Ca2q-free normoxic medium. After 5 min, the medium was switched to the Ca2q-free ischemia-like medium. The effect of procaine was evaluated by adding procaine to each medium. 2.5. Statistical analysis The data obtained from the histology were evaluated with the Kruskal–Wallis test followed by the Mann–Whitney test. The data obtained by measuring the DC potential were analyzed by analysis of variance followed by Dunnett’s test. The data from the fluorometry were analyzed using a repeated two-way analysis of variance to detect differences among groups. When differences were found, Scheffe’s ´ test was used post hoc to compare each value with that in the control group.
3. Results Figs. 1 and 2 show the extent of neuronal damage in the hippocampal CA1 region in each group. In the control animals, almost all pyramidal cells were degenerated 7
Fig. 3. The typical change in the DC potential shift produced by 3 min of transient forebrain ischemia.
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Table 1 Effects of procaine on the anoxic depolarization Onset Žs. Control 53"16 Procaine 0.2 m mol 71"13 0.4 m mol 79"14) 2.0 m mol 102"27))
Amplitude ŽmV. Recovery Žs. Duration Žs. 22"11
101"39
206"53
19"10 19"7 22"10
94"32 117"51 116"44
176"37 193"61 164"50
The differences in the DC potential shift were compared regarding its onset latency, amplitude, recovery time of the depolarization to half-maximal amplitude, and the duration of the half-maximal amplitude. ) p0.05, )) p- 0.01 compared with the respective values in the control group.
days after ischemia. The number of preserved neurons was 8 " 6 Žmean " S.D., n s 10.. In contrast, the varying doses of procaine reduced the damage significantly. The numbers of preserved neurons were 53 " 23 Žprocaine 0.2 m mol., 181 " 35 Žprocaine 0.4 m mol., and 246 " 14 Žprocaine 2 m mol., and there were no differences between the both sides. In the animals that received 2 m mol of procaine, almost all pyramidal cells remained intact 7 days after ischemia. However, in 8 of these 10 gerbils, the respiration stopped for about 1 min after the intracerebroventricular administration of procaine Ž2 m mol., and the animals started breathing spontaneously after about 1 min. As shown in Fig. 3, the forebrain ischemia provoked a gradual decrease in the extracellular membrane potential in the hippocampal CA1 area. Then, the DC potential shifted suddenly. After reperfusion, the membrane repolarized gradually. In the animals that received procaine Ž0.2, 0.4, or 2 m mol., the onset latencies were prolonged by 37%, 49%, and 100%, respectively, compared with the control group ŽTable 1.. However, the maximal amplitude of the DC potential shift, the recovery time of the depolarization, and the duration of the half-maximal amplitude were not significantly different among all groups. Fig. 4 is comprised of photographs showing the elevation of the wCa2q x i in hippocampal slices induced by the in vitro ischemia-like condition. As shown in Fig. 5, when the hippocampal slice was perfused with the in vitro ischemia-like medium, almost no increase in the ratio of the wCa2q x i was observed in the CA1 field within 250 s after the beginning of the in vitro hypoxia. Subsequently, an acute and large increase in the wCa2q x i spread throughout the CA1 field, and the ratio reached a plateau. When slices were perfused with the procaine-containing medium, the onset and the extent of the wCa2 x i increase were dose-dependently inhibited with the effect being significant at the procaine concentrations of 50 and 100 m molrl. As shown in Fig. 6, when the slices were perfused with Ca2q-free in vitro ischemia-like medium, an increase in the wCa2q x i was observed in the CA1 field in a manner similar to that in the Ca2q-containing condition. Perfusion with procaine Ž100 m molrl.-containing medium produced a
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Fig. 4. Photographs showing the difference between a control slice and a procaine Ž100 m molrl.-perfusing slice in the wCa2q x i elevation induced by in vitro hypoxia. ŽA. 250 s, ŽB. 300 s, ŽC. 350 s, ŽD. 400 s, and ŽE. 450 s after the start of hypoxia. The open rectangles represent the measured areas. Bar s 0.5 mm.
J. Chen et al.r Brain Research 792 (1998) 16–23
Fig. 5. Changes in the ratio of the wCa2q x i in slices of the gerbil hippocampal CA1 field which underwent in vitro ischemia-like conditions. The standard ischemia-like medium Ž`., procaine Ž10 m molrl.containing ischemia-like medium Žv ., procaine Ž50 m molrl.-containing ischemia-like medium Ž'., and procaine Ž100 m molrl.-containing ischemia-like medium ŽB. effects are shown. Each value represents the mean"S.D. of 10 slices. ) p- 0.05, )) p- 0.01, ))) p- 0.001 compared with the respective values in the standard ischemia-like group.
Fig. 6. Changes in the ratio of wCa2q x i in slices of the hippocampal CA1 field which underwent Ca2q-free in vitro ischemia-like conditions. The Ca2q-free in vitro ischemia-like medium Ž`., and procaine Ž100 m molrl.-containing Ca2q-free in vitro ischemia-like medium Žv . effects are shown. Each value represents the mean"S.D. of 8 or 10 slices. ) p- 0.05, )) p- 0.01, ))) p- 0.001 compared with the respective values in the Ca2q-free ischemia-like group.
gradual and moderate increase in the wCa2q x i . The latency at the beginning of the increase in the wCa2q x i was markedly prolonged, and the extent of the increase was also depressed by 100 m molrl of procaine.
4. Discussion In the present study, we observed the improvement of the ischemia-induced damage in hippocampal CA1 pyramidal cells and a prolongation of the onset of the AD in the in vivo experiments. We also observed the inhibition of the hypoxia-induced increase in the wCa2q x i by procaine in the in vitro experiments. The brain temperature was carefully maintained at 37.58C in all experiments, because the brain temperature in ischemia and reperfusion plays an important role in the histologic outcome w15x. Under this normothermic condi-
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tion, transient forebrain ischemia for 3 min produced a marked damage in the hippocampal CA1 region in control animals, which is consistent with a previous report w15x. In contrast, the intracerebroventricular administration of procaine dose-dependently ameliorated the neuronal damage, although a high dose of procaine Ž2 m mol. inhibited the spontaneous respiration in animals. The hippocampal CA1 area is vulnerable to ischemia, and this region is innervated by glutamatergic fibers w24x, which cause neuronal death by their excess release of glutamate w9x. The excess release of glutamate in ischemia has been shown to be caused mainly by the reversal of the Naq-cotransport system in a Ca2q-independent manner w20x, and the blockade of Naq channels by tetrodotoxin or lidocaine reduces the Ca2q-independent excitatory amino acid release w5,13,25,27x. Therefore, the blockade of Naq channels by procaine may take part in the inhibition of the release of excitatory amino acid, thereby reducing the glutamate toxicity. The Naq gradient across the cell membrane is an energy source for Naq–Ca2q and Naq–Kq exchange as well as glutamate uptake from the extracellular space. The Naq–Kq ATPase pump uses ATP to maintain the Naq gradient. In the early period of ischemia, the function of the ion pump is maintained by the cellular storage of ATP. When ATP is depleted, the ion pump loses its function and various neurotransmitters are released, which cause a sudden depolarization of the membrane w5,7,17,23x. In the present experiment, we found that the intracerebroventricular administration of procaine prolonged the onset latency of the sudden shift of the DC potential. The protective effect of this agent may be due to the prolongation of the onset of the AD. The inhibition of the Naq influx through Naq channels by procaine may block the initiation of the catastrophic enzymatic process leading to neuronal damage. It has been generally believed that an excessive increase in the wCa2q x i leads to irreversible neuronal injury w4,10x. In our present in vitro experiment, we found a sudden and large increase in the wCa2q x i in the CA1 area in the ischemia-like condition. This finding is in good agreement with the selective vulnerability in this area. The extracellular DC potential shift closely reflects the movement of Naq, Kq, Cly and Ca2q across the membrane w7x, and the acute and large negative shift of the membrane potential has been shown to be accompanied by an acute elevation of the wCa2q x i w17x. Therefore, the sudden shift of the AD observed in the in vivo experiment is closely related to the sudden increase in the wCa2q x i in the in vitro experiment. For these reasons, the protective effects of procaine in the in vivo experiment are thought to have been caused by the prevention of the increase in the wCa2q x i . It has been demonstrated that a large elevation of the wCa2q x i induced by cerebral ischemia in the CA1 field of the hippocampus was caused by both the Ca2q influx from the extracellular space and the Ca2q release from the
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intracellular Ca2q stores such as the endoplasmic reticulum and mitochondria w16x. A similar phenomenon was observed in the present study. The wCa2q x i increased in the Ca2q-free hypoxic condition as well as in the Ca2q-containing condition, although the extent of the increase was smaller than that in the Ca2q-containing condition. Therefore, it is speculated that the Ca2q release from intracellular stores plays an important role in the elevation of the wCa2q x i in ischemia, as does the influx of Ca2q from the extracellular space. There are two mechanisms, by which Ca2q is released from the endoplasmic reticulum to the cytosol w8,26x. One is the release through ryanodine receptors, which exist on the membrane of the endoplasmic reticulum. The increase in the cytosolic concentration of Ca2q, which is caused by the influx of Ca2q from the extracellular space through mainly N-methyl-D-aspartate ŽNMDA.-gated Ca2q channels in an ischemic event, stimulates the release of Ca2q from the endoplasmic reticulum by activating ryanodine receptors ŽCa2q-induced Ca2q release.. The other mechanism involves the inositol 1,4,5-triphosphate ŽIP3 . receptors on the endoplasmic reticulum. The stimulation of postsynaptic metabotropic glutamate receptors activates the intracellular phosphatidylinositol ŽPI. turnover. Thus, IP3 receptor-linked Ca2q channels open in response to the increase in the intracellular concentration of IP3 , resulting in the release of Ca2q ŽIP3-induced Ca2q release.. The mechanism by which procaine reduces the release of Ca2q from the intracellular store was not elucidated in the present study. However, procaine has been shown to be a blocker of ryanodine receptors, and to inhibit ryanodinemediated Ca2q release from the endoplasmic reticulum. Procaine has also been demonstrated not to affect IP3mediated Ca2q release w14,21,29x. Therefore, procaine may inhibit the ryanodine-mediated Ca2q release in ischemia. In this study, the protective effects of procaine on hippocampal CA1 pyramidal cells against ischemia by inhibiting the onset time of the AD and preventing the increase in the wCa2q x i were observed. The reduction of the wCa2q x i in the early stage of ischemia seems to play an important role in preventing the cascade leading to neuronal damage, and the present findings contribute to the clarification of the neuroprotective action of procaine against ischemia-induced neuronal damage.
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