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Guanidinoethane sulfate is neuroprotective towards delayed CA1 neuronal death in gerbils
Life Sciences, Vol. 56, No. 14, pp. 1201-1206, 1995 Copyright 0 1995 Ekvier Science Ltd Printed in the USA. All rights MC& Km-3205/95 $9.50 t .OO
Pergamon 0024-3205(95)00059-3
GUANIDINOETHANE SULFATE IS NEUROPROTECTIVE TOWARDS DELAYED CA1 NEURONAL DEATH IN GERBILS Hironaka Igarashi*@, Ingrid L. Kwee@, and Tsutomu Nakada$@ Neurochemistry Department
Research Laboratory, VANCSC, Martinez, CA 94553$ and of Neurology, University of California, Davis, CA 95616” (Received in final form January 10, 1995)
Summarv The potential neuroprotective effects of guanidinoethane sulfate (GES) on delayed neuronal death of hippocampal CA1 neurons were investigated using a gerbil model of forebrain ischemia. Neuronal densities of CA, neurons in the saline control group (255.1 * 11.7 cells/mm) and guanidinoethane sulfate pretreated control group (249.0 + 9.4 cells/mm) showed no significant differences. By contrast, in animals subjected to ischemia, CA, neurons of the guanidinoethane sulfate pretreated group showed a significantly higher number of surviving neurons (61.1 % 55.11 cells/mm) compared to the saline group (17.75 f 12.73 cells/mm) (p < 0.05, t-test). The study indicated that although partial, guanidinoethane sulfate is neuroprotective towards gerbil hippocampal CA1 neurons against ischemic insult. Key Words: guanidinoethane sulfate, neuroprotection,
CA, neurons
Guanidinoethane sulfate (GES) is a taurine analogue originally introduced by Huxtable et al. as competitive inhibitor of taurine transport into the brain (1,2). Recent studies in our laboratory disclosed that GES is also an effective alkaline shifter of itttrnce/hrInr brain pH and can significantly reduce the extent of brain itt/rncel/tr/~r lactic acidosis brought about by anoxic insult in mice (3). Further studies demonstrated that pretreatment of mice with GES can significantly enhance the survival rate of mice exposed to fatal doses of anoxia only if GES accumulation in brain is sufficiently high to produce a significant brain alkaline shift (4). The findings suggested a potential role of GES, by effecting an alkali shift, on cellular protection against anoxia/ischemia. In this study, we extended our investigation to the effects of GES pretreatment on delayed neuronal death of hippocampal CA1 neurons using a gerbil forebrain ischemia model. Methods Fxperiniettld
Des@
Adult male gerbils (ca. 70 g) were divided into two groups, saline and GES groups. The GES group received daily injections of GES (dissolved in distilled water and neutralized to pH = 7.0 with IN NaOH), 0.25 ml (625 mg/kg), intraperitoneally for two weeks, while the saline group Tsutomu Nakada, M.D., Ph.D, Dcpartmcnt
Muir Road, Martinez, CA 94553
of Neurology, University of California, Davis, VANCSC, 150
1202
GES Protects CA, Neurons against Ischemia
Vol. 56, No. 14, 1995
received sham saline injections (normal saline). This GES regimen has been determined to produce sufficient GES accumulation to effect an alkali shift without significantly affecting physiological parameters such as blood pressure and blood gas measurements (3). Seven animals from each group were utilized for baseline GES and taurine assay and brain pH determination by 31P nuclear magnetic resonance (NMR) spectroscopy. A separate set of animals from each group was used for the ischemia studies described below.
Taurine and methyl GES was prepared as described by Huxtable et al. (2). isothiopseudourea were purchased from Sigma (St. Louis, MO). Taurine (60g) was mixed with methyl isothiopseudourea ( IOOg) in concentrated ammonium hydroxide (120ml) under a chemical hood. The mixture was heated to 60°C and maintained at this temperature until evolution of gas ceased. (Note: methane thiol is generated as Huxtable et al. warned!) The solution was cooled on ice to crystalize GES, and filtered. The crystal was dissolved in a minimal volume of distilled water and recrystalized three times from water. The purity of GES was confirmed by proton NMR spectroscopy at 500 MHz (GE Omega-SOO).
Animals were sacrificed by exsanguination under pentobarbital anesthesia. Brains were removed and fixed in liquid nitrogen The frozen brain was pulverized in a liquid nitrogen cooled mortar and pestle, and mixed with powdered frozen perchloric acid, 0.5 N, 4 volumes. The powder mixture was centritiged in liquid nitrogen cooled centrifuge tubes at 16,000 rpm at -4°C for 30 minutes. The supernatant was removed and titrated to a pH of 7.0-7.5 by the addition of potassium bicarbonate. The solution was stored in an ice bath for 15 minutes and the potassium perchlorate precipitate was removed by centrifugation at 16,000 rpm at -4°C. For GES assay (2), aliquots (0.5 ml) of the extracts obtained were placed on a dual bed ion exchange column containing 6 x 0.5 cm of Dowex AGI anion exchange resin (chloride form) layered over 6 x 0.5cm of Dowex AG50 cation exchange resin (hydrogen form). Water (3ml) was then added to the column to collect the eluent, GES, free from other naturally occurring guanidino compounds, such as arginine. The color reagent was 5% w/v naphthol in ethanol containing 2.5% of 1% aqueous diacetyl solution. Aliquots of eluent were taken and diluted to 0.7ml with water. Sodium hydroxide (3N; 0. lml) was added followed by 0.2 ml of the color reagent. After 18 min, absorbance was read at 545 nm using a Gilford Spectrophotometer (Model 2600). A Varian LC 5000 HPLC system with Gilson Microfractionator and EM ScienceLiChrospher column (SI 100 RP-8, 10 micron, 10 mm x 25 cm) was used for taurine assay. An aliquot of the extract prepared as above, ca. 2 ml, was passed through a cation-exchange column (Bio-Rad Laboratories, AG 5OW-X8, 200-400 mesh, H+ form, 5 x 15 mm) and washed three times with 1 ml of distilled water. The final volume of the eluate was ca. 5ml. Pre-column treatment was necessary to eliminate interfering substances and help protect the column from contamination caused by too much absorption of other amino acids. Taurine was labelled with dansyl chloride (DNS-Cl) as follows. Taurine sample, 0.2 ml, was combined with DNS-Cl, 0.2 ml, and heated in a water bath at 40°C for ten minutes to complete the labelling reaction. 10 ~1 of the sample was injected into the column and eluted at a flow rate of 0.5 ml/min with a mobile phase of methanol:water (36:65 v/v), 0.6% acetic acid and 0.008% triethylamine. Taurine concentration was determined at an absorbance of 254nm.
Vol. 56, No. 14, 19%
GES Protects CA, Neurons against Ischemia
1203
Brain pH was determined norl-itwn.~il~ely using 31P NMR spectroscopy. A GE Omega CSI7T (clear bore 120 mm, horizontal, 7T) was used. Animals were anesthetized with pentobarbital, 35 mg/kg, intraperitoneally. Animals were held in a padded holder which was placed in the probe chamber containing a one turn round surface coil (5 mm diameter) tunable to the resonance frequency of proton and 31P. To avoid signal contamination, the scalp and temporal muscle were retracted. The animal body temperature was kept at 37*0.5”C using a non-magnetic heating pad. Field homogeneity was maximized by shimming on tissue water proton signals. 31P spectra were obtained using a one pulse sequence at 121.67 MHz with a spectra1 width of 10K and stored into 4K memory blocks (recycle time 2.7 set). The broad resonance with short T2 on 31P spectra was Line broadening of 30 Hz was applied as removed using the convolution difference technique. noise filter. Intracellular pH was estimated using the equation: pH=6,77+log{(S-3.29)/(5.68-S)}, where 6 is the chemical shift of inorganic phosphate (Pi) referred to that of phosphocreatine (PCr).
The gerbil forebrain ischemia model of Kirino was employed (5). Animals were anesthetized with 1% halothane, 70% nitrous oxide, and 29% oxygen. The temporal muscle and recta1 temperature were monitored and kept rigorously constant at ca. 37.1 “C using a controlled heating system starting 30 minutes prior to one hour after induction of ischemia. Ischemia in experimental groups (saline and GES ischemia groups) was induced by occlusion of the carotid arteries bilaterally for 5 minutes using Sugita aneurysm clips, Animals in the sham control groups (saline and GES controls) received sham surgery.
After recirculation of seven days, animals were given transcardial perfusion with 50 cc of normal saline followed by 500 cc of 5% paraformaldehyde in O.lM phosphate buffer (pH=7.3), at 100 cm H,O, under deep pentobarbital anesthesia. Brains were removed and fixed in the same buffered paraformaldehyde solution for 14 days. Six pm thick sections containing the dorsal hippocampus at the level 1.4-1.7 mm posterior to the bregma were prepared on a cryostat from paraffin embedded brain. Sections were stained with hematoxylin and eosin and examined by two experienced personnel in a double blinded fashion. The number of viable neurons within the CA, region was counted using an Olympus BH-2 microscope at x 400. The results represent the average of the counts of the two examiners. Results Brain GES and taurine levels and intracelllular pH are summarized in TABLE I. The degree of taurine replacement by GES is confirmed to be sufficient to produce brain pH alkali shift (3). His/oiogy Representative photomicrographs of the control (A), saline ischemia (B), and GES ischemia (C) groups are shown in Figure 1. TABLE 11 summarizes the CA1 neuronal counts. There were no significant differences in neuronal density between saline control and GES control groups indicating that GES pretreatment did not have deleterious effects on neuronal density in CA1 region. In contrast, significantly higher numbers of neurons were preserved in the GES ischemia group than in the saline ischemia group confirming the neuroprotective effects of GES.
Vol. 56, No. 14, 1995
GES Protects CA, Neurons against Ischemia
1204
Brain GES/Taurine
Saline group (n=7) GES group (n=7)
TABLE I Levels and Intracellular
pH brain pH
taurine (pmol/g wet weight) 9.77 * 0.60 5.90 f 0.12
GES (pmol/g wet weight) co.01 2.37kO.36
7.17 f 0.03* > 7.20
* mean f standard deviation
C
B
A
Fig. I Representative photomicrographs for (A) control group, (B) saline ischemia group, and (C) GES ischemia group, x 400. Note the significantly higher neuronal density in the GES ischemia group compared to the saline ischemia group.
TABLE II Neuronal Density in CA, region (/mm) saline control GES control saline ischemia GES &hernia
group group group group
(n=5) (n=5) (n=lO) (n=16)
1 255.1 k 11.7’5 1 249.0 f 9.4§ 17.75 f 12.73’ 1 61.1 f 55.11’
Smean and standard deviation §NS, *p < 0.05 (t-test)
Vol. 56, No. 14, 1995
GES Protects CA, Neurons against Ischemia
12.05
Discussion The study clearly demonstrated that GES is neuroprotective towards gerbil hippocampal CA1 neurons against delayed death following forebrain ischemia. Currently, two effects of GES pretreatment on brain are known, namely, reduction of taurine level and pH alkaline shift. The present study was designed to judge whether or not GES pretreatment indeed confers protective effects towards hippocampal CA, neurons and was not designed to investigate the underlying mechanism of GES protection. Nevertheless, it is of interest to speculate about it. Two potential cellular effects of taurine which may play a role in anoxic/ischemic brain injury are the concept that: (a) taurine may serve as an inhibitory neurotransmitter; and (b) taurine may serve as an intracellular scavenger of free radicals. Both effects suggest that taurine itself may be protective towards anoxic/ischemic brain injury. GES reduces rather than increases taurine levels in rat brain, including hippocampus (6). Therefore, theoretically, GES pretreatment should be harmful rather than protecive. However, in contrast, our study demonstrated that pretreatment with GES and reduction of taurine results in a protective effect on hippocampal CA, neurons. Lehmann et al. (6) has also demonstrated in their kainate toxicity studies that the neuronal loss caused by kainate injection into the hippocampus was not modified by GES pretreatment. The study concluded that the decline in taurine has neither sensitizing nor protective effect on rat hippocampal neurons against the metabolic and toxic effects of kainate. Therefore, it is plausible that the effects of GES on brain taurine levels per se are unlikely to be the main factor for GES protection on CA, neurons shown in this study. Several lines of evidence support the concept that the severity of cerebra1 lactic acidosis is an important factor in determining the outcome of brain anoxia/ischemia (7-l 1). Although the precise role of acidosis in delayed neuronal loss remains to be elucidated, it is conceivable that, as one of the earliest biochemical sequelae of anoxic/ischemic insult, acidosis may play a role as trigger of deleterious biochemicalbiophysical cascades which lead to delayed damage in hippocampal CAi neurons. Indeed, Munekata and Hossmann have demonstrated that post-ischemic acidosis is more pronounced in the hippocampal CA, region of the gerbil indicating that degree of acidosis may play a role in the selective vulnerability of delayed neuronal death (I 2). It is plausible that the observed alkali shift brought about by GES may indeed represent a significant, if not main, factor subserving the mechanism of GES protection shown in this study. Interestingly, hypothermia, which is well known to be neuroprotective to CA1 neurons in delayed neuronal death, is also known to produce an alkaline shift in brain pH (13,14). In summary, the study demonstrated that GES is neuroprotective towards delayed neuronal loss in hippocampal CA, neuron. Although precise mechanism by which GES conveys neuroprotection remains to be elucidated, existing literature suggests the brain pH alkali shift brought about by GES pretreatment may play a role. Further investigation on the neuroprotective effects of GES, especially in brain pH regulation, is strongly warranted. Acknowledgements The study was supported by Grants from the U. S. Public Health Service (GM 37197, RR 025 1 l), Department of Veterans Affairs Research Service, and Nihon Medi-Physics, Inc. References 1. R. J. HUXTABLE and S. E. LIPPINCOTT, Arch. Biochem. Biophys. 210 698709 (1981). 2. R. J. HUXTABLE, H. E. LAIRD II, and S. E. LIPPINCOTT, J. Pharmacol. Exp. Therap. 211 465-471 (1979).
1206
3. 4. 5. 6.
GES Protects CA, Neurons against Ischemia
Vol. 56, No. 14, 1995
T. NAKADA and I. L. KWEE, NeuroRepot-t $1035-1038 (1993). T. NAKADA and I. L. KWEE, Sot. Neurosci. Abstr., p. 1078 (1991). T. KIRINO and A. TAMURA, Stroke 17 455-459 (1986). A. LEHMANN, H. HAGBERG, R. J. HUXTABLE, and M. SANDBERG, Neurochem. Int. lo 265-274 (1987). 7. J. H. HALSEY, K. A. CONGER, A. G. HIJDETZ, F. M. G. HOBBES, J. H. GARCIA, and E. R. STRONG, Neurol. Res. lo 97-104 (1988). 8. R. E. MYERS and S. YAMAGUCHI, Arch. Neurol. 34 65-74 (1977). 9. F. PLUM, Neurology 33 222-233 (1983). 10. F. A. WELSH, M. D. GINSBERG, W. RIEDER, and W. W. BUDD, Stroke u 355-363 (1980). 11, E. WHITE, H. J. JUCHNIEWICZ, and J. B. CLARK,. J. Neurochem. 52 154-161 (1989). 12. K. MUNEKATA and K. A. HOSSMANN,. Stroke 18 412-417 (1987). 13. D. C. JOHNSON, M. NISHIMURA, P. OKUNIFF, H. KAZEMI, and B. HITZIG, J. Appl. Physiol. 67 2527-2534 (1989). 14. A. R. LAPTOOK, R. J. T. CORBETT, R. STERETT, D. BURNS, G. TOLLEFSBOL, and D. GARCIA, Sot. Magn. Reson. Med Abstr., p. 520 (1993).
Pergamon 0024-3205(95)00059-3
GUANIDINOETHANE SULFATE IS NEUROPROTECTIVE TOWARDS DELAYED CA1 NEURONAL DEATH IN GERBILS Hironaka Igarashi*@, Ingrid L. Kwee@, and Tsutomu Nakada$@ Neurochemistry Department
Research Laboratory, VANCSC, Martinez, CA 94553$ and of Neurology, University of California, Davis, CA 95616” (Received in final form January 10, 1995)
Summarv The potential neuroprotective effects of guanidinoethane sulfate (GES) on delayed neuronal death of hippocampal CA1 neurons were investigated using a gerbil model of forebrain ischemia. Neuronal densities of CA, neurons in the saline control group (255.1 * 11.7 cells/mm) and guanidinoethane sulfate pretreated control group (249.0 + 9.4 cells/mm) showed no significant differences. By contrast, in animals subjected to ischemia, CA, neurons of the guanidinoethane sulfate pretreated group showed a significantly higher number of surviving neurons (61.1 % 55.11 cells/mm) compared to the saline group (17.75 f 12.73 cells/mm) (p < 0.05, t-test). The study indicated that although partial, guanidinoethane sulfate is neuroprotective towards gerbil hippocampal CA1 neurons against ischemic insult. Key Words: guanidinoethane sulfate, neuroprotection,
CA, neurons
Guanidinoethane sulfate (GES) is a taurine analogue originally introduced by Huxtable et al. as competitive inhibitor of taurine transport into the brain (1,2). Recent studies in our laboratory disclosed that GES is also an effective alkaline shifter of itttrnce/hrInr brain pH and can significantly reduce the extent of brain itt/rncel/tr/~r lactic acidosis brought about by anoxic insult in mice (3). Further studies demonstrated that pretreatment of mice with GES can significantly enhance the survival rate of mice exposed to fatal doses of anoxia only if GES accumulation in brain is sufficiently high to produce a significant brain alkaline shift (4). The findings suggested a potential role of GES, by effecting an alkali shift, on cellular protection against anoxia/ischemia. In this study, we extended our investigation to the effects of GES pretreatment on delayed neuronal death of hippocampal CA1 neurons using a gerbil forebrain ischemia model. Methods Fxperiniettld
Des@
Adult male gerbils (ca. 70 g) were divided into two groups, saline and GES groups. The GES group received daily injections of GES (dissolved in distilled water and neutralized to pH = 7.0 with IN NaOH), 0.25 ml (625 mg/kg), intraperitoneally for two weeks, while the saline group Tsutomu Nakada, M.D., Ph.D, Dcpartmcnt
Muir Road, Martinez, CA 94553
of Neurology, University of California, Davis, VANCSC, 150
1202
GES Protects CA, Neurons against Ischemia
Vol. 56, No. 14, 1995
received sham saline injections (normal saline). This GES regimen has been determined to produce sufficient GES accumulation to effect an alkali shift without significantly affecting physiological parameters such as blood pressure and blood gas measurements (3). Seven animals from each group were utilized for baseline GES and taurine assay and brain pH determination by 31P nuclear magnetic resonance (NMR) spectroscopy. A separate set of animals from each group was used for the ischemia studies described below.
Taurine and methyl GES was prepared as described by Huxtable et al. (2). isothiopseudourea were purchased from Sigma (St. Louis, MO). Taurine (60g) was mixed with methyl isothiopseudourea ( IOOg) in concentrated ammonium hydroxide (120ml) under a chemical hood. The mixture was heated to 60°C and maintained at this temperature until evolution of gas ceased. (Note: methane thiol is generated as Huxtable et al. warned!) The solution was cooled on ice to crystalize GES, and filtered. The crystal was dissolved in a minimal volume of distilled water and recrystalized three times from water. The purity of GES was confirmed by proton NMR spectroscopy at 500 MHz (GE Omega-SOO).
Animals were sacrificed by exsanguination under pentobarbital anesthesia. Brains were removed and fixed in liquid nitrogen The frozen brain was pulverized in a liquid nitrogen cooled mortar and pestle, and mixed with powdered frozen perchloric acid, 0.5 N, 4 volumes. The powder mixture was centritiged in liquid nitrogen cooled centrifuge tubes at 16,000 rpm at -4°C for 30 minutes. The supernatant was removed and titrated to a pH of 7.0-7.5 by the addition of potassium bicarbonate. The solution was stored in an ice bath for 15 minutes and the potassium perchlorate precipitate was removed by centrifugation at 16,000 rpm at -4°C. For GES assay (2), aliquots (0.5 ml) of the extracts obtained were placed on a dual bed ion exchange column containing 6 x 0.5 cm of Dowex AGI anion exchange resin (chloride form) layered over 6 x 0.5cm of Dowex AG50 cation exchange resin (hydrogen form). Water (3ml) was then added to the column to collect the eluent, GES, free from other naturally occurring guanidino compounds, such as arginine. The color reagent was 5% w/v naphthol in ethanol containing 2.5% of 1% aqueous diacetyl solution. Aliquots of eluent were taken and diluted to 0.7ml with water. Sodium hydroxide (3N; 0. lml) was added followed by 0.2 ml of the color reagent. After 18 min, absorbance was read at 545 nm using a Gilford Spectrophotometer (Model 2600). A Varian LC 5000 HPLC system with Gilson Microfractionator and EM ScienceLiChrospher column (SI 100 RP-8, 10 micron, 10 mm x 25 cm) was used for taurine assay. An aliquot of the extract prepared as above, ca. 2 ml, was passed through a cation-exchange column (Bio-Rad Laboratories, AG 5OW-X8, 200-400 mesh, H+ form, 5 x 15 mm) and washed three times with 1 ml of distilled water. The final volume of the eluate was ca. 5ml. Pre-column treatment was necessary to eliminate interfering substances and help protect the column from contamination caused by too much absorption of other amino acids. Taurine was labelled with dansyl chloride (DNS-Cl) as follows. Taurine sample, 0.2 ml, was combined with DNS-Cl, 0.2 ml, and heated in a water bath at 40°C for ten minutes to complete the labelling reaction. 10 ~1 of the sample was injected into the column and eluted at a flow rate of 0.5 ml/min with a mobile phase of methanol:water (36:65 v/v), 0.6% acetic acid and 0.008% triethylamine. Taurine concentration was determined at an absorbance of 254nm.
Vol. 56, No. 14, 19%
GES Protects CA, Neurons against Ischemia
1203
Brain pH was determined norl-itwn.~il~ely using 31P NMR spectroscopy. A GE Omega CSI7T (clear bore 120 mm, horizontal, 7T) was used. Animals were anesthetized with pentobarbital, 35 mg/kg, intraperitoneally. Animals were held in a padded holder which was placed in the probe chamber containing a one turn round surface coil (5 mm diameter) tunable to the resonance frequency of proton and 31P. To avoid signal contamination, the scalp and temporal muscle were retracted. The animal body temperature was kept at 37*0.5”C using a non-magnetic heating pad. Field homogeneity was maximized by shimming on tissue water proton signals. 31P spectra were obtained using a one pulse sequence at 121.67 MHz with a spectra1 width of 10K and stored into 4K memory blocks (recycle time 2.7 set). The broad resonance with short T2 on 31P spectra was Line broadening of 30 Hz was applied as removed using the convolution difference technique. noise filter. Intracellular pH was estimated using the equation: pH=6,77+log{(S-3.29)/(5.68-S)}, where 6 is the chemical shift of inorganic phosphate (Pi) referred to that of phosphocreatine (PCr).
The gerbil forebrain ischemia model of Kirino was employed (5). Animals were anesthetized with 1% halothane, 70% nitrous oxide, and 29% oxygen. The temporal muscle and recta1 temperature were monitored and kept rigorously constant at ca. 37.1 “C using a controlled heating system starting 30 minutes prior to one hour after induction of ischemia. Ischemia in experimental groups (saline and GES ischemia groups) was induced by occlusion of the carotid arteries bilaterally for 5 minutes using Sugita aneurysm clips, Animals in the sham control groups (saline and GES controls) received sham surgery.
After recirculation of seven days, animals were given transcardial perfusion with 50 cc of normal saline followed by 500 cc of 5% paraformaldehyde in O.lM phosphate buffer (pH=7.3), at 100 cm H,O, under deep pentobarbital anesthesia. Brains were removed and fixed in the same buffered paraformaldehyde solution for 14 days. Six pm thick sections containing the dorsal hippocampus at the level 1.4-1.7 mm posterior to the bregma were prepared on a cryostat from paraffin embedded brain. Sections were stained with hematoxylin and eosin and examined by two experienced personnel in a double blinded fashion. The number of viable neurons within the CA, region was counted using an Olympus BH-2 microscope at x 400. The results represent the average of the counts of the two examiners. Results Brain GES and taurine levels and intracelllular pH are summarized in TABLE I. The degree of taurine replacement by GES is confirmed to be sufficient to produce brain pH alkali shift (3). His/oiogy Representative photomicrographs of the control (A), saline ischemia (B), and GES ischemia (C) groups are shown in Figure 1. TABLE 11 summarizes the CA1 neuronal counts. There were no significant differences in neuronal density between saline control and GES control groups indicating that GES pretreatment did not have deleterious effects on neuronal density in CA1 region. In contrast, significantly higher numbers of neurons were preserved in the GES ischemia group than in the saline ischemia group confirming the neuroprotective effects of GES.
Vol. 56, No. 14, 1995
GES Protects CA, Neurons against Ischemia
1204
Brain GES/Taurine
Saline group (n=7) GES group (n=7)
TABLE I Levels and Intracellular
pH brain pH
taurine (pmol/g wet weight) 9.77 * 0.60 5.90 f 0.12
GES (pmol/g wet weight) co.01 2.37kO.36
7.17 f 0.03* > 7.20
* mean f standard deviation
C
B
A
Fig. I Representative photomicrographs for (A) control group, (B) saline ischemia group, and (C) GES ischemia group, x 400. Note the significantly higher neuronal density in the GES ischemia group compared to the saline ischemia group.
TABLE II Neuronal Density in CA, region (/mm) saline control GES control saline ischemia GES &hernia
group group group group
(n=5) (n=5) (n=lO) (n=16)
1 255.1 k 11.7’5 1 249.0 f 9.4§ 17.75 f 12.73’ 1 61.1 f 55.11’
Smean and standard deviation §NS, *p < 0.05 (t-test)
Vol. 56, No. 14, 1995
GES Protects CA, Neurons against Ischemia
12.05
Discussion The study clearly demonstrated that GES is neuroprotective towards gerbil hippocampal CA1 neurons against delayed death following forebrain ischemia. Currently, two effects of GES pretreatment on brain are known, namely, reduction of taurine level and pH alkaline shift. The present study was designed to judge whether or not GES pretreatment indeed confers protective effects towards hippocampal CA, neurons and was not designed to investigate the underlying mechanism of GES protection. Nevertheless, it is of interest to speculate about it. Two potential cellular effects of taurine which may play a role in anoxic/ischemic brain injury are the concept that: (a) taurine may serve as an inhibitory neurotransmitter; and (b) taurine may serve as an intracellular scavenger of free radicals. Both effects suggest that taurine itself may be protective towards anoxic/ischemic brain injury. GES reduces rather than increases taurine levels in rat brain, including hippocampus (6). Therefore, theoretically, GES pretreatment should be harmful rather than protecive. However, in contrast, our study demonstrated that pretreatment with GES and reduction of taurine results in a protective effect on hippocampal CA, neurons. Lehmann et al. (6) has also demonstrated in their kainate toxicity studies that the neuronal loss caused by kainate injection into the hippocampus was not modified by GES pretreatment. The study concluded that the decline in taurine has neither sensitizing nor protective effect on rat hippocampal neurons against the metabolic and toxic effects of kainate. Therefore, it is plausible that the effects of GES on brain taurine levels per se are unlikely to be the main factor for GES protection on CA, neurons shown in this study. Several lines of evidence support the concept that the severity of cerebra1 lactic acidosis is an important factor in determining the outcome of brain anoxia/ischemia (7-l 1). Although the precise role of acidosis in delayed neuronal loss remains to be elucidated, it is conceivable that, as one of the earliest biochemical sequelae of anoxic/ischemic insult, acidosis may play a role as trigger of deleterious biochemicalbiophysical cascades which lead to delayed damage in hippocampal CAi neurons. Indeed, Munekata and Hossmann have demonstrated that post-ischemic acidosis is more pronounced in the hippocampal CA, region of the gerbil indicating that degree of acidosis may play a role in the selective vulnerability of delayed neuronal death (I 2). It is plausible that the observed alkali shift brought about by GES may indeed represent a significant, if not main, factor subserving the mechanism of GES protection shown in this study. Interestingly, hypothermia, which is well known to be neuroprotective to CA1 neurons in delayed neuronal death, is also known to produce an alkaline shift in brain pH (13,14). In summary, the study demonstrated that GES is neuroprotective towards delayed neuronal loss in hippocampal CA, neuron. Although precise mechanism by which GES conveys neuroprotection remains to be elucidated, existing literature suggests the brain pH alkali shift brought about by GES pretreatment may play a role. Further investigation on the neuroprotective effects of GES, especially in brain pH regulation, is strongly warranted. Acknowledgements The study was supported by Grants from the U. S. Public Health Service (GM 37197, RR 025 1 l), Department of Veterans Affairs Research Service, and Nihon Medi-Physics, Inc. References 1. R. J. HUXTABLE and S. E. LIPPINCOTT, Arch. Biochem. Biophys. 210 698709 (1981). 2. R. J. HUXTABLE, H. E. LAIRD II, and S. E. LIPPINCOTT, J. Pharmacol. Exp. Therap. 211 465-471 (1979).
1206
3. 4. 5. 6.
GES Protects CA, Neurons against Ischemia
Vol. 56, No. 14, 1995
T. NAKADA and I. L. KWEE, NeuroRepot-t $1035-1038 (1993). T. NAKADA and I. L. KWEE, Sot. Neurosci. Abstr., p. 1078 (1991). T. KIRINO and A. TAMURA, Stroke 17 455-459 (1986). A. LEHMANN, H. HAGBERG, R. J. HUXTABLE, and M. SANDBERG, Neurochem. Int. lo 265-274 (1987). 7. J. H. HALSEY, K. A. CONGER, A. G. HIJDETZ, F. M. G. HOBBES, J. H. GARCIA, and E. R. STRONG, Neurol. Res. lo 97-104 (1988). 8. R. E. MYERS and S. YAMAGUCHI, Arch. Neurol. 34 65-74 (1977). 9. F. PLUM, Neurology 33 222-233 (1983). 10. F. A. WELSH, M. D. GINSBERG, W. RIEDER, and W. W. BUDD, Stroke u 355-363 (1980). 11, E. WHITE, H. J. JUCHNIEWICZ, and J. B. CLARK,. J. Neurochem. 52 154-161 (1989). 12. K. MUNEKATA and K. A. HOSSMANN,. Stroke 18 412-417 (1987). 13. D. C. JOHNSON, M. NISHIMURA, P. OKUNIFF, H. KAZEMI, and B. HITZIG, J. Appl. Physiol. 67 2527-2534 (1989). 14. A. R. LAPTOOK, R. J. T. CORBETT, R. STERETT, D. BURNS, G. TOLLEFSBOL, and D. GARCIA, Sot. Magn. Reson. Med Abstr., p. 520 (1993).