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Dimethylsulfoxide (DMSO) treatment reduces infarction volume after permanent focal cerebral ischemia in rats
Neuroscience Letters 239 (1997) 125–127
Dimethylsulfoxide (DMSO) treatment reduces infarction volume after permanent focal cerebral ischemia in rats Shigetoshi Shimizu a, Roger P. Simon a, Steven H. Graham a , b ,* a
Department of Neurology, University of Pittsburgh, S-526 Biomedical Science Tower, Pittsburgh, PA 15261, USA b Neurology Service, University Drive, Veterans Affairs Medical Center, S-526 Biomedical Science Tower, Pittsburgh, PA 15261, USA Received 3 October 1997; received in revised form 18 November 1997; accepted 18 November 1997
Abstract Dimethylsulfoxide (DMSO) is a common vehicle used for many drugs used in neuroprotective experiments. DMSO has many biological effects, including antiinflammatory, antioxidant, and local anesthetic effects that could be neuroprotective. To determine if DMSO is neuroprotective in ischemia, DMSO (0, 0.01, 0.03, 0.1, 0.3 and 1.0 ml) was administered intraperitoneally 30 min prior to permanent middle cerebral artery (MCA) occlusion in the rat. Twenty-four hours after MCA occlusion, brains were removed and sectioned. Mean infarction volume was significantly reduced in rats treated with 0.1, 0.3 and 1.0 ml of DMSO compared to saline controls. There was no acute effect of drug treatment upon arterial blood gasses or mean blood pressure. These results suggest that DMSO is neuroprotective in focal cerebral ischemia. Investigators must use appropriate controls when DMSO is used as a vehicle. 1997 Elsevier Science Ireland Ltd.
Keywords: Dimethylsulfoxide; Cerebral ischemia
Dimethylsulfoxide (DMSO) is a simple organic compound that has a plethora of biological actions including antioxidant, antiinflammatory, antinociceptive, and radioprotective effects [2,7]. DMSO is highly soluble and distributes throughout the entire body including brain very rapidly [11]. Furthermore, it solubilizes many lipophilic compounds and increases penetrance of many drugs into brain. Accordingly, it is frequently used as a vehicle in pharmacological studies in cerebral ischemia. Central to many of its biological actions is its free radical scavenging activity. DMSO traps the hydroxide radical and dimethylsulfide, the metabolite of DMSO, traps oxygen free radicals [21]. Since many antioxidant compounds may be neuroprotective [4], we investigated whether DMSO itself is neuroprotective in a rat model of stroke. Permanent focal ischemia was induced by the intraarterial suture method, as initially described by Longa et al. [13] with modifications. All studies were performed under a pro* Corresponding author. Tel.: +1 412 6483299; fax: +1 412 6481239; e-mail: [email protected]
tocol approved by the University of Pittsburgh Animal Care and Use Committee. Briefly, 275–300 g male Sprague– Dawley rats were anesthetized with 4% isoflurane (70% N2O/30% O2) and incubated. Then the rats were ventilated with 1.5% isoflurane (70% N2O/30%O2). Rectal temperature was kept at 37 ± 0.5°C with a heating pad, and left temporalis muscle temperature was maintained at 37.5 ± 0.2°C with a heating lamp. One femoral artery was cannulated to monitor blood pressure, arterial blood gases, and blood glucose. The external carotid artery was ligated with a 6-0 silk suture and dissected distally, and the internal carotid artery (ICA) was isolated and separated from the vagus nerve. The extracranial branch of the ICA was ligated close to its origin with a 6-0 silk suture. A 3-0 monofilament nylon suture was introduced into the ICA lumen through the stump of the external carotid artery and advanced 20–22 mm past the common carotid artery bifurcation. DMSO (Sigma, St. Louis, MO, USA) was mixed with saline to total volume 1.0 ml. For the dose-response study, doses of 0.01, 0.03, 0.1, 0.3 and 1.0 ml were administered intraperitoneally 30 min prior to permanent MCA occlusion.
0304-3940/97/$17.00 1997 Elsevier Science Ireland Ltd. All rights reserved PII S0304- 3940(97) 00915- 4
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S. Shimizu et al. / Neuroscience Letters 239 (1997) 125–127
Fig. 1. Mean infarction volume determined from TTC-stained sections 24 h after permanent MCA occlusion. Error bars represent SD. *P , 0.05 less than saline controls.
The wound was sutured, anesthesia discontinued and the rats returned to their cages. The suture was left in place until the rats were sacrificed 24 h after onset of ischemia. The rats were anesthetized, decapitated and their brains rapidly removed and sectioned coronally at 2 mm intervals. Sections were immersed in 2% 2,3,5-triphenyltetrazolim hydrochloride (TTC) in saline for 20 min at 37°C then transferred to 4% paraformaldehyde for 15 min. Six sections were analyzed for infarction size by blinded observer using computerized image analysis system (MCID, St Catharines, Ontario, Canada). Infarction area in each section was calculated by subtracting the normal area that stained with TTC in the right ischemic hemisphere from area of the left nonischemic hemisphere [18]. Data were expressed as the mean ± SD. Infarction volumes and physiological variables were statistically analyzed by one-way factorial analysis of variance followed by Fisher’s PLSD multiple comparison test. P , 0.05 was regarded as significant. Fig. 1 summarizes the changes in mean infarct volume. There was a significant (P , 0.05) reduction in infarction volume in rats treated with 0.1, 0.3 and 1.0 ml DMSO. Although there was a tread toward reduction in infarction volume, 0.01 and 0.03 ml did not significantly reduce infarction volume. There was no significant effect of DMSO treatment upon mean arterial pressure or arterial blood gases (Table 1). DMSO has a number of actions that may account for its
neuroprotective effects. It is a free radical scavenger and thus may reduce oxidative stress after ischemia. DMSO may also inhibit prostaglandin receptors [16]. DMSO has local anesthetic effects and thus could ameliorate ischemic injury by effects on cell excitation [17]. DMSO may also have vasodilatory effects [10], although blood pressure effects were not observed acutely in this study. A number of studies suggested that DMSO is useful for the treatment of central nervous system injuries. DMSO was effective in reducing increased intracranial pressure, but not brain water content in cold-lesion induced cerebral edema in rabbits [8]. DMSO treatment improved blood flow antioxidative metabolism in a model of rhesus monkey missile brain injury [3]. DMSO treatment reduced mortality rate in a model of brain compression in rhesus monkeys [5]. Furthermore, DMSO treatment reduced hippocampal cell death after injection of the excitotoxin beta-N-oxalylamino-l-alanine [20]. Previous experiments in other models of stroke have produced contradictory results. There was a suggestion of less severe histopathologic changes in DMSO-treated rhesus monkeys after MCA occlusion; however, only two monkeys per group were analyzed [6]. DMSO treatment reduced infarction size in a canine MCA embolectomy model [12]. However in the gerbil carotid occlusion DMSO treatment reduced blood–brain barrier permeability, but increased infarction size compared to saline controls [15]. The human use of DMSO was popular two decades ago when the compound was used for many inflammatory disorders. Clinical trials have suggested that it was as effective in the treatment of intracranial hypertension in brain trauma patients, but may produce significant side effects including fluid and electrolyte disorders [9,19]. Since the compound is a readily available chemical, it was widely used without prescription for treatment of inflammatory disorders. However, it became apparent that DMSO may be toxic especially when used chronically: DMSO may produce many side effects for local skin irritation to intravascular hemolysis [7]. Accordingly, its human use has been curtailed. There are several lines of evidence that oxidative stress contributes to secondary damage after cerebral ischemia. Superoxide radicals, perhydroxyl and hydrogen peroxide radicals are produced after ischemia [4]. Ischemic damage is exacerbated when antioxidative defense systems such as copper-zinc superoxide dismutase are overexpressed [22].
Table 1 Mean± SD of arterial blood pressure (MABP), arterial blood gasses and plasma glucose obtained 10 min after onset of ischemia MABP (mmHg) Saline (ml) 0.01 0.03 0.01 0.3 1.0
110 108 105 107 106 110
± ± ± ± ± ±
8 12 13 8 10 10
PaO2 (mmHg) 132 125 124 127 124 130
± ± ± ± ± ±
11 11 12 15 17 13
PaCO2 (mmHg) 37 38 36 35 35 34
± ± ± ± ± ±
3 3 3 2 1 1
pH 7.41 7.39 7.39 7.39 7.41 7.41
Glucose (mg/dl) ± ± ± ± ± ±
0.03 0.03 0.04 0.02 0.02 0.03
136 144 139 135 129 141
± ± ± ± ± ±
12 31 14 27 30 28
S. Shimizu et al. / Neuroscience Letters 239 (1997) 125–127
Free radical scavengers such as dimethylthiourea and tirilazad mesylate have been shown to reduce infarction volume after MCA occlusion [1,14]. The current results demonstrate that DMSO in doses of 0.1 ml and greater significantly reduce infarction volume. Thus, DMSO should be used cautiously and with appropriate controls in cerebral ischemia experiments. This work was supported in part by NIH NS 24728 (R.P.S.) and the Department of Veterans Affairs Merit Review Program (S.H.G.). We thank Marie Rose for technical support and Pat Strickler for secretarial support. [1] Andrus, P.K., Taylor, B.M., Sun, F.F. and Hall, E.D., Effects of the lipid peroxidation inhibitor tirilazad mesylate (U-74006F) on gerbil brain eicosanoid levels following ischemia and reperfusion, Brain Res., 659 (1994) 126–132. [2] Ashwood-Smith, M.J., Radioprotective and cryoprotective properties of dimethyl sulfoxide in cellular systems, Ann. N. Y. Acad. Sci., 141 (1967) 45–62. [3] Brown, F.D., Johns, L.M. and Mullan, S., Dimethyl sulfoxide in experimental brain injury, with comparison to mannitol, J. Neurosurg., 53 (1980) 58–62. [4] Chan, P.H., Role of oxidants in ischemic brain damage, Stroke, 27 (1996) 1124–1129. [5] de la Torre, J.C., Rowed, D.W., Kawanaga, H.M. and Mullan, S., Dimethyl sulfoxide in the treatment of experimental brain compression, J. Neurosurg., 38 (1973) 345–354. [6] de la Torre, J.C. and Surgeon, J.W., Dexamethasone and DMSO in experimental transorbital cerebral infarction, Stroke, 7 (1976) 577–583. [7] Jacob, S.W. and Wood, D.C., Dimethyl sulfoxide (DMSO). Toxicology, pharmacology, and clinical experience, Am. J. Surg., 114 (1967) 414–426. [8] James, H.E., Camp, P.E., Harbaugh, R.D., Marshall, L.F. and Werner, R., Comparison of the effects of DMSO and pentobarbitone on experimental brain oedema, Acta Neurochir. (Wien), 60 (1982) 245–255. [9] Karaca, M., Bilgin, U.Y., Akar, M. and de la Torre, J.C., Dimethyl sulfoxide lowers ICP after closed head trauma, Eur. J. Clin. Pharmacol., 40 (1991) 113–114. [10] Klingman, A.M., Topical pharmacology of dimethyl sulfoxide (DMSO), J. Am. Med. Assoc., 193 (1965) 796–802.
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[11] Kolb, K.H., Jaenicke, G., Kramer, M. and Schulze, P.E., Absorption, distribution and elimination of labeled dimethyl sulfoxide in man and animals, Ann. N. Y. Acad. Sci., 141 (1967) 85–95. [12] Laha, R.K., Dujovny, M., Barrionuevo, P.J., DeCastro, S.C., Hellstrom, H.R. and Maroon, J.C., Protective effects of methyl prednisolone and dimethyl sulfoxide in experimental middle cerebral artery embolectomy, J. Neurosurg., 49 (1978) 508– 516. [13] Longa, E.Z., Weinstein, P.R., Carlson, S. and Cummins, R., Reversible middle cerebral artery occlusion without craniectomy in rats, Stroke, 20 (1989) 84–91. [14] Martz, D., Rayos, G., Schielke, G.P. and Betz, A.L., Allopurinol and dimethylthiourea reduce brain infarction following middle cerebral artery occlusion in rats, Stroke, 20 (1989) 488– 494. [15] McGraw, C.P., The effect of dimethyl sulfoxide (DMSO) on cerebral infarction in the Mongolian gerbil, Acta Neurol. Scand. Suppl., 64 (1977) 160–161. [16] Rao, C.V., Differential effects of detergents and dimethylsulfoxide on membrane prostaglandin E1 and F2 alpha receptors, Life Sci., 20 (1977) 2013–2022. [17] Shealy, C.N., The physiological substrate of pain, Headache, 6 (1966) 101–108. [18] Swanson, R.A., Morton, M.T., Tsao-Wu, G., Savalos, R.A., Davidson, C. and Sharp, F.R., A semiautomated method for measuring brain infarct volume, J. Cerebral Blood Flow Metab., 10 (1990) 290–293. [19] Waller, F.T., Tanabe, C.T. and Paxton, H.D., Treatment of elevated intracranial pressure with dimethyl sulfoxide, Ann. N. Y. Acad. Sci., 411 (1983) 286–292. [20] Willis, C.L., Meldrum, B.S., Nunn, P.B., Anderton, B.H. and Leigh, P.N., Neuroprotective effect of free radical scavengers on beta-N-oxalylamino-l-alanine (BOAA)-induced neuronal damage in rat hippocampus, Neurosci. Lett., 182 (1994) 159– 162. [21] Wood, D.C. and Wood, J., Pharmacologic and biochemical considerations of dimethyl sulfoxide, Ann. N. Y. Acad. Sci., 243 (1975) 7–19. [22] Yang, G., Chan, P.H., Chen, J., Carlson, E., Chen, S.F., Weinstein, P., Epstein, C.J. and Kamii, H., Human copperzinc superoxide dismutase transgenic mice are highly resistant to reperfusion injury after focal cerebral ischemia, Stroke, 25 (1994) 165–170.
Dimethylsulfoxide (DMSO) treatment reduces infarction volume after permanent focal cerebral ischemia in rats Shigetoshi Shimizu a, Roger P. Simon a, Steven H. Graham a , b ,* a
Department of Neurology, University of Pittsburgh, S-526 Biomedical Science Tower, Pittsburgh, PA 15261, USA b Neurology Service, University Drive, Veterans Affairs Medical Center, S-526 Biomedical Science Tower, Pittsburgh, PA 15261, USA Received 3 October 1997; received in revised form 18 November 1997; accepted 18 November 1997
Abstract Dimethylsulfoxide (DMSO) is a common vehicle used for many drugs used in neuroprotective experiments. DMSO has many biological effects, including antiinflammatory, antioxidant, and local anesthetic effects that could be neuroprotective. To determine if DMSO is neuroprotective in ischemia, DMSO (0, 0.01, 0.03, 0.1, 0.3 and 1.0 ml) was administered intraperitoneally 30 min prior to permanent middle cerebral artery (MCA) occlusion in the rat. Twenty-four hours after MCA occlusion, brains were removed and sectioned. Mean infarction volume was significantly reduced in rats treated with 0.1, 0.3 and 1.0 ml of DMSO compared to saline controls. There was no acute effect of drug treatment upon arterial blood gasses or mean blood pressure. These results suggest that DMSO is neuroprotective in focal cerebral ischemia. Investigators must use appropriate controls when DMSO is used as a vehicle. 1997 Elsevier Science Ireland Ltd.
Keywords: Dimethylsulfoxide; Cerebral ischemia
Dimethylsulfoxide (DMSO) is a simple organic compound that has a plethora of biological actions including antioxidant, antiinflammatory, antinociceptive, and radioprotective effects [2,7]. DMSO is highly soluble and distributes throughout the entire body including brain very rapidly [11]. Furthermore, it solubilizes many lipophilic compounds and increases penetrance of many drugs into brain. Accordingly, it is frequently used as a vehicle in pharmacological studies in cerebral ischemia. Central to many of its biological actions is its free radical scavenging activity. DMSO traps the hydroxide radical and dimethylsulfide, the metabolite of DMSO, traps oxygen free radicals [21]. Since many antioxidant compounds may be neuroprotective [4], we investigated whether DMSO itself is neuroprotective in a rat model of stroke. Permanent focal ischemia was induced by the intraarterial suture method, as initially described by Longa et al. [13] with modifications. All studies were performed under a pro* Corresponding author. Tel.: +1 412 6483299; fax: +1 412 6481239; e-mail: [email protected]
tocol approved by the University of Pittsburgh Animal Care and Use Committee. Briefly, 275–300 g male Sprague– Dawley rats were anesthetized with 4% isoflurane (70% N2O/30% O2) and incubated. Then the rats were ventilated with 1.5% isoflurane (70% N2O/30%O2). Rectal temperature was kept at 37 ± 0.5°C with a heating pad, and left temporalis muscle temperature was maintained at 37.5 ± 0.2°C with a heating lamp. One femoral artery was cannulated to monitor blood pressure, arterial blood gases, and blood glucose. The external carotid artery was ligated with a 6-0 silk suture and dissected distally, and the internal carotid artery (ICA) was isolated and separated from the vagus nerve. The extracranial branch of the ICA was ligated close to its origin with a 6-0 silk suture. A 3-0 monofilament nylon suture was introduced into the ICA lumen through the stump of the external carotid artery and advanced 20–22 mm past the common carotid artery bifurcation. DMSO (Sigma, St. Louis, MO, USA) was mixed with saline to total volume 1.0 ml. For the dose-response study, doses of 0.01, 0.03, 0.1, 0.3 and 1.0 ml were administered intraperitoneally 30 min prior to permanent MCA occlusion.
0304-3940/97/$17.00 1997 Elsevier Science Ireland Ltd. All rights reserved PII S0304- 3940(97) 00915- 4
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S. Shimizu et al. / Neuroscience Letters 239 (1997) 125–127
Fig. 1. Mean infarction volume determined from TTC-stained sections 24 h after permanent MCA occlusion. Error bars represent SD. *P , 0.05 less than saline controls.
The wound was sutured, anesthesia discontinued and the rats returned to their cages. The suture was left in place until the rats were sacrificed 24 h after onset of ischemia. The rats were anesthetized, decapitated and their brains rapidly removed and sectioned coronally at 2 mm intervals. Sections were immersed in 2% 2,3,5-triphenyltetrazolim hydrochloride (TTC) in saline for 20 min at 37°C then transferred to 4% paraformaldehyde for 15 min. Six sections were analyzed for infarction size by blinded observer using computerized image analysis system (MCID, St Catharines, Ontario, Canada). Infarction area in each section was calculated by subtracting the normal area that stained with TTC in the right ischemic hemisphere from area of the left nonischemic hemisphere [18]. Data were expressed as the mean ± SD. Infarction volumes and physiological variables were statistically analyzed by one-way factorial analysis of variance followed by Fisher’s PLSD multiple comparison test. P , 0.05 was regarded as significant. Fig. 1 summarizes the changes in mean infarct volume. There was a significant (P , 0.05) reduction in infarction volume in rats treated with 0.1, 0.3 and 1.0 ml DMSO. Although there was a tread toward reduction in infarction volume, 0.01 and 0.03 ml did not significantly reduce infarction volume. There was no significant effect of DMSO treatment upon mean arterial pressure or arterial blood gases (Table 1). DMSO has a number of actions that may account for its
neuroprotective effects. It is a free radical scavenger and thus may reduce oxidative stress after ischemia. DMSO may also inhibit prostaglandin receptors [16]. DMSO has local anesthetic effects and thus could ameliorate ischemic injury by effects on cell excitation [17]. DMSO may also have vasodilatory effects [10], although blood pressure effects were not observed acutely in this study. A number of studies suggested that DMSO is useful for the treatment of central nervous system injuries. DMSO was effective in reducing increased intracranial pressure, but not brain water content in cold-lesion induced cerebral edema in rabbits [8]. DMSO treatment improved blood flow antioxidative metabolism in a model of rhesus monkey missile brain injury [3]. DMSO treatment reduced mortality rate in a model of brain compression in rhesus monkeys [5]. Furthermore, DMSO treatment reduced hippocampal cell death after injection of the excitotoxin beta-N-oxalylamino-l-alanine [20]. Previous experiments in other models of stroke have produced contradictory results. There was a suggestion of less severe histopathologic changes in DMSO-treated rhesus monkeys after MCA occlusion; however, only two monkeys per group were analyzed [6]. DMSO treatment reduced infarction size in a canine MCA embolectomy model [12]. However in the gerbil carotid occlusion DMSO treatment reduced blood–brain barrier permeability, but increased infarction size compared to saline controls [15]. The human use of DMSO was popular two decades ago when the compound was used for many inflammatory disorders. Clinical trials have suggested that it was as effective in the treatment of intracranial hypertension in brain trauma patients, but may produce significant side effects including fluid and electrolyte disorders [9,19]. Since the compound is a readily available chemical, it was widely used without prescription for treatment of inflammatory disorders. However, it became apparent that DMSO may be toxic especially when used chronically: DMSO may produce many side effects for local skin irritation to intravascular hemolysis [7]. Accordingly, its human use has been curtailed. There are several lines of evidence that oxidative stress contributes to secondary damage after cerebral ischemia. Superoxide radicals, perhydroxyl and hydrogen peroxide radicals are produced after ischemia [4]. Ischemic damage is exacerbated when antioxidative defense systems such as copper-zinc superoxide dismutase are overexpressed [22].
Table 1 Mean± SD of arterial blood pressure (MABP), arterial blood gasses and plasma glucose obtained 10 min after onset of ischemia MABP (mmHg) Saline (ml) 0.01 0.03 0.01 0.3 1.0
110 108 105 107 106 110
± ± ± ± ± ±
8 12 13 8 10 10
PaO2 (mmHg) 132 125 124 127 124 130
± ± ± ± ± ±
11 11 12 15 17 13
PaCO2 (mmHg) 37 38 36 35 35 34
± ± ± ± ± ±
3 3 3 2 1 1
pH 7.41 7.39 7.39 7.39 7.41 7.41
Glucose (mg/dl) ± ± ± ± ± ±
0.03 0.03 0.04 0.02 0.02 0.03
136 144 139 135 129 141
± ± ± ± ± ±
12 31 14 27 30 28
S. Shimizu et al. / Neuroscience Letters 239 (1997) 125–127
Free radical scavengers such as dimethylthiourea and tirilazad mesylate have been shown to reduce infarction volume after MCA occlusion [1,14]. The current results demonstrate that DMSO in doses of 0.1 ml and greater significantly reduce infarction volume. Thus, DMSO should be used cautiously and with appropriate controls in cerebral ischemia experiments. This work was supported in part by NIH NS 24728 (R.P.S.) and the Department of Veterans Affairs Merit Review Program (S.H.G.). We thank Marie Rose for technical support and Pat Strickler for secretarial support. [1] Andrus, P.K., Taylor, B.M., Sun, F.F. and Hall, E.D., Effects of the lipid peroxidation inhibitor tirilazad mesylate (U-74006F) on gerbil brain eicosanoid levels following ischemia and reperfusion, Brain Res., 659 (1994) 126–132. [2] Ashwood-Smith, M.J., Radioprotective and cryoprotective properties of dimethyl sulfoxide in cellular systems, Ann. N. Y. Acad. Sci., 141 (1967) 45–62. [3] Brown, F.D., Johns, L.M. and Mullan, S., Dimethyl sulfoxide in experimental brain injury, with comparison to mannitol, J. Neurosurg., 53 (1980) 58–62. [4] Chan, P.H., Role of oxidants in ischemic brain damage, Stroke, 27 (1996) 1124–1129. [5] de la Torre, J.C., Rowed, D.W., Kawanaga, H.M. and Mullan, S., Dimethyl sulfoxide in the treatment of experimental brain compression, J. Neurosurg., 38 (1973) 345–354. [6] de la Torre, J.C. and Surgeon, J.W., Dexamethasone and DMSO in experimental transorbital cerebral infarction, Stroke, 7 (1976) 577–583. [7] Jacob, S.W. and Wood, D.C., Dimethyl sulfoxide (DMSO). Toxicology, pharmacology, and clinical experience, Am. J. Surg., 114 (1967) 414–426. [8] James, H.E., Camp, P.E., Harbaugh, R.D., Marshall, L.F. and Werner, R., Comparison of the effects of DMSO and pentobarbitone on experimental brain oedema, Acta Neurochir. (Wien), 60 (1982) 245–255. [9] Karaca, M., Bilgin, U.Y., Akar, M. and de la Torre, J.C., Dimethyl sulfoxide lowers ICP after closed head trauma, Eur. J. Clin. Pharmacol., 40 (1991) 113–114. [10] Klingman, A.M., Topical pharmacology of dimethyl sulfoxide (DMSO), J. Am. Med. Assoc., 193 (1965) 796–802.
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[11] Kolb, K.H., Jaenicke, G., Kramer, M. and Schulze, P.E., Absorption, distribution and elimination of labeled dimethyl sulfoxide in man and animals, Ann. N. Y. Acad. Sci., 141 (1967) 85–95. [12] Laha, R.K., Dujovny, M., Barrionuevo, P.J., DeCastro, S.C., Hellstrom, H.R. and Maroon, J.C., Protective effects of methyl prednisolone and dimethyl sulfoxide in experimental middle cerebral artery embolectomy, J. Neurosurg., 49 (1978) 508– 516. [13] Longa, E.Z., Weinstein, P.R., Carlson, S. and Cummins, R., Reversible middle cerebral artery occlusion without craniectomy in rats, Stroke, 20 (1989) 84–91. [14] Martz, D., Rayos, G., Schielke, G.P. and Betz, A.L., Allopurinol and dimethylthiourea reduce brain infarction following middle cerebral artery occlusion in rats, Stroke, 20 (1989) 488– 494. [15] McGraw, C.P., The effect of dimethyl sulfoxide (DMSO) on cerebral infarction in the Mongolian gerbil, Acta Neurol. Scand. Suppl., 64 (1977) 160–161. [16] Rao, C.V., Differential effects of detergents and dimethylsulfoxide on membrane prostaglandin E1 and F2 alpha receptors, Life Sci., 20 (1977) 2013–2022. [17] Shealy, C.N., The physiological substrate of pain, Headache, 6 (1966) 101–108. [18] Swanson, R.A., Morton, M.T., Tsao-Wu, G., Savalos, R.A., Davidson, C. and Sharp, F.R., A semiautomated method for measuring brain infarct volume, J. Cerebral Blood Flow Metab., 10 (1990) 290–293. [19] Waller, F.T., Tanabe, C.T. and Paxton, H.D., Treatment of elevated intracranial pressure with dimethyl sulfoxide, Ann. N. Y. Acad. Sci., 411 (1983) 286–292. [20] Willis, C.L., Meldrum, B.S., Nunn, P.B., Anderton, B.H. and Leigh, P.N., Neuroprotective effect of free radical scavengers on beta-N-oxalylamino-l-alanine (BOAA)-induced neuronal damage in rat hippocampus, Neurosci. Lett., 182 (1994) 159– 162. [21] Wood, D.C. and Wood, J., Pharmacologic and biochemical considerations of dimethyl sulfoxide, Ann. N. Y. Acad. Sci., 243 (1975) 7–19. [22] Yang, G., Chan, P.H., Chen, J., Carlson, E., Chen, S.F., Weinstein, P., Epstein, C.J. and Kamii, H., Human copperzinc superoxide dismutase transgenic mice are highly resistant to reperfusion injury after focal cerebral ischemia, Stroke, 25 (1994) 165–170.