- Email: [email protected]
Hypoxia preconditioning attenuates brain edema associated with kainic acid-induced status epilepticus in rats
Brain Research 825 Ž1999. 189–193
Short communication
Hypoxia preconditioning attenuates brain edema associated with kainic acid-induced status epilepticus in rats Mitchell R. Emerson b
a,b
, Stanley R. Nelson b , Fred E. Samson b , Thomas L. Pazdernik
a,b,)
a Department of Pharmacology, Toxicology, and Therapeutics, UniÕersity of Kansas Medical Center, Kansas City, KS 66160-7417, USA Smith Mental Retardation and Human DeÕelopment Research Center, UniÕersity of Kansas Medical Center, Kansas City, KS 66160-7417, USA
Accepted 26 January 1999
Abstract Kainic acid ŽKA.-induced seizures elicit edema associated with necrosis in susceptible brain regions Že.g., piriform cortex and hippocampal CA 1 and CA 3 regions.. To test the hypothesis that hypoxia preconditioning protects against KA-induced edema formation, adult male rats were exposed to a 9% O 2 , 91% N2 atmosphere for 8 h. KA Ž14 mgrkg, i.p.. was administered 1, 3, 7, or 14 days later. Regional analysis of edema indicated that hypoxia exposure attenuated edema formation in piriform and frontal cortices and hippocampus when KA was given 1, 3, or 7 days later but not 14 days after hypoxia. Cycloheximide Ž2 mgrkg s.c.. given 1 h prior to hypoxia prevented the protective effect of hypoxia on KA-induced edema attenuation in the piriform cortex and hippocampus. Thus, hypoxic challenge induces a general adaptive response that protects against the seizure-associated pathophysiology, with no direct relationship to seizure intensity. This response may involve stress-related transcription factors and effector proteins. q 1999 Elsevier Science B.V. All rights reserved. Keywords: Hypoxia preconditioning; Kainic acid; Status epilepticus; Seizure; Brain edema; Specific gravity
Hypoxiarischemia preconditioning paradigms can produce neuroprotection against subsequent brain insults. This phenomenon is based on the premise that a sublethal hypoxicrischemic episode will afford neuroprotection against later challenges even of a different kind w8x. The biochemical changes responsible for the preconditioning response are purported to include the stress-related proteins Žheat shock proteins w12x, superoxide dismutases w7x., as well as adenosine release andror receptor activation w4x. Systemic injection of kainic acid ŽKA. into rats causes a glutamate-driven status epilepticus that leads to a cellular calcium influx and oxidative stress similar to that seen in ischemia-reperfusion injury w13,15x. An early cytotoxic edema and a late vasogenic edema associated with necrosis occur in brain regions Ži.e., piriform cortex and hippocampal CA 1 and CA 3 regions. extensively involved in seizure activity w2,18x. KA augments the release of glutamate from neurons located in the hippocampus and piriform cortex w9,19x. Excess stimulation of glutamatergic pathways increases intracellular Naq and Ca2q concentrations leading
)
Corresponding [email protected]
author.
Fax:
q 1-913-588-5677;
E-mail:
to cytotoxic osmotic swelling of neurons and glia. As neuronal damage progresses to necrosis, a vasogenic edema results from reduced integrity of vessel endothelial walls and collapse of the blood–brain barrier w18x. Pohle and Rauca w14x report that exposure to mild hypoxia decreases the incidence of KA-induced seizures and thus lethality, as well as the KA-associated neuropathology when challenge is given 1 week later. The purpose of our study was to examine the effects of hypoxia preconditioning on KA-induced seizures and subsequent brain edema. The extent of protection as a function of the interval between hypoxia and KA challenge and the requirement for protein synthesis was studied. Adult male Wistar rats Ž250–350 g; Harlan Sprague– Dawley, Indianapolis, IN. were kept under standard conditions including a 12-h lightrdark cycle with access to food and water ad libitum. Rats were exposed to either a mild hypoxic Ž9% O 2 , 91% N2 . atmosphere ŽPuritan-Bennett, Kansas City, KS. in a flow-through chamber or to normal laboratory air for 8 h during the dark cycle. At timepoints of 1, 3, 7, or 14 days later, rats were injected with 0.9% saline as control or KA Ž14 mgrkg i.p.. dissolved in 0.9% saline. Twenty-four hours after KA or saline administration, rats were anesthetized with halothane, decapitated
0006-8993r99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved. PII: S 0 0 0 6 - 8 9 9 3 Ž 9 9 . 0 1 1 9 5 - 6
190
M.R. Emerson et al.r Brain Research 825 (1999) 189–193
and brains were removed for specific gravity measurements. For the protein synthesis studies, cycloheximide Ž2 mgrkg s.c.. dissolved in 0.9% saline was given 1 h prior to the 8 h of hypoxia or laboratory air exposure. Immediately after removal, brains were dissected under cold Ž48C. kerosene. Specific gravities were determined on samples about 2 mm3 in volume from brain regions of interest using a density gradient column consisting of kerosene and bromobenzene w11x. In edematous brain re-
gions, a decrease in specific gravity of brain tissue occurs as a result of the influx of fluid w11x. The percent tissue volume change was calculated from the specific gravity by the equation: % change in tissue volume as water s
Ž sp. gr.y 1 . cont.r Ž sp. gr.y 1 . exp. 4 y 1 = 100
where: sp. gr.s specific gravity; cont.s control; and exp.s experimental.
Fig. 1. Temporal effect of hypoxia preconditioning on KA-induced edema Žspecific gravity reduction. in various brain regions. ŽA. Hypoxia exposure prevented the formation of edema in the piriform cortex when KA challenge was given 1, 3, or 7 days later. Increases in tissue volume due to influx of water were detected following KA alone Ž9.58 " 1.71%. and KA-14 days post-hypoxia Ž24.54 " 2.02%.. ŽB. Hypoxia exposure prevented the formation of edema in the frontal cortex when KA challenge was given 1, 3, or 7 days later. Increases in tissue volume due to influx of water were detected following KA alone Ž6.27 " 2.10%. and KA-14 days post-hypoxia Ž18.45 " 8.50%.. ŽC. Hypoxia exposure prevented the formation of edema in the hippocampus when KA challenge was given 1, 3, or 7 days later. Increases in tissue volume due to influx of water were detected following KA alone Ž2.23 " 0.92%. and KA-14 days post-hypoxia Ž5.24 " 2.65%.. ŽD. The cerebellar vermis is considered non-responsive to KA, however, a decrease in tissue volume in the KA-1 day post-hypoxia group Žy4.65 " 1.85%. and an increase in the KA-7 days post-hypoxia group Ž1.78 " 1.13%. were detected possibly representing the heterogeneity of sampling in this tissue. All columns and bars represent the mean" S.E.M. Saline Ž n s 12.; KA Ž n s 29.; 1 day Ž n s 5.; 3 days Ž n s 10.; 7 days Ž n s 11.; and 14 days Ž n s 4.. U P - 0.05, significantly different from KA ŽKruskal–Wallis..
M.R. Emerson et al.r Brain Research 825 (1999) 189–193
Statistical significance was determined using a nonparametric analysis of variance procedure ŽKruskal–Wallis.. This procedure was used since our graphical analysis did not provide evidence that our data was distributed normally with constant variance. Immediately following hypoxia, animals appeared in good condition with the exception of hyperventilation and less exploratory activity than rats kept outside of the hypoxia chamber. These behaviors ceased within minutes of removal from the chamber. Rats exposed to hypoxia lost almost 4 g of weight, indicating a mild stress. Non-exposed rats gained approximately 9 g, a normal weight gain during the dark cycle. Hypoxia-exposed rats regained lost
191
weight within 24–48 h and continued weight gain at a normal rate when compared to non-exposed rats Ždata not shown.. Twenty-four hours after KA-administration, rats lost approximately 10% of their body weight. There was no difference in weight loss following KA in rats that were exposed to hypoxia, and those that were not Ždata not shown.. After KA challenge, there was a decrease in the specific gravity values Ži.e., increased edema. in piriform and frontal cortices, and hippocampus, but not cerebellum. In the three affected regions, the specific gravity decreases were considerably less in animals exposed to hypoxia and injected with KA 1, 3, or 7 days later. On the other hand,
Fig. 2. Effect of CHX given 1 h prior to hypoxia preconditioning on KA-induced edema Žspecific gravity reduction. in various brain regions when KA challenge is given 7 days later ŽCHX-Hypox. vs. hypoxia preconditioning alone when KA challenge is given 7 days later ŽHypox.. ŽA. CHX reversed hypoxia’s attenuation of edema formation in the piriform cortex. ŽB. CHX had no effect on hypoxia’s attenuation of edema formation in the frontal cortex. ŽC. CHX reversed hypoxia’s attenuation of edema formation in the hippocampus. ŽD. CHX caused an increase in edema formation in the cerebellar vermis. All columns and bars represent the mean " S.E.M. Saline Ž n s 9.; KA Ž n s 29.; Hypox Ž n s 11.; CHX-Hypox Ž n s 8.. U P - 0.05, significantly different from Hypox ŽKruskal–Wallis..
192
M.R. Emerson et al.r Brain Research 825 (1999) 189–193
animals challenged with KA 14 days following hypoxia exposure were not protected against the development of edema in the brain regions studied ŽFig. 1.. Cycloheximide given 1 h prior to hypoxia exposure significantly reversed the protection in the hippocampus and piriform cortex when the KA challenge was given 7 days later. The response in the frontal cortex was not significantly altered by cycloheximide pretreatment ŽFig. 2.. Hypoxia preconditioning protected against brain damage from KA-induced seizures assessed by specific gravity measurements of edema in susceptible brain regions. Hypoxia produced a mild stress as evident by hyperventilation, decreased exploratory behavioral activity and a modest weight loss. This mild stress initiates neurochemical changes, some of which require protein synthesis, that confer neuroprotection when rats are given a subsequent challenge such as KA-induced seizures. Pohle and Rauca w14x previously reported that exposure to mild hypoxia for 8 h provides neuroprotection against KA-induced histopathologic damage when the challenge was given 7 days later, primarily through reduction in seizure intensity. In our studies, the protection against edema formation was relatively short-lived Ži.e., 1 day through 7 days, absent at 14 days. and required protein synthesis in the limbic brain regions. However, there was no difference among groups in seizure intensity when lethality was included in the scoring system. Thus, it is notable that protection did occur in animals with intense but not lethal seizures. An adaptive response may be a contributing factor in protection conferred by hypoxia preconditioning. The stress response includes an alteration in gene expression induced by adverse conditions, such as ischemia or excitotoxicity w10x. Stress-related genes are activated under a variety of conditions and their effector proteins may participate in the protective response to KA-induced seizures. Some of these include the heat shock proteins, superoxide dismutases, metallothioneins, and heme oxygenases w7,10,12x. Which genes are altered during a specific response will depend on the nature and severity of the insult Ži.e., which transcription factors are inducedrrepressed during and after the insult.. To examine the dependence on protein synthesis in the neuroprotective effect, Ži.e., gene expression of protective proteins. rats were pretreated with cycloheximide 1 h prior to hypoxia exposure, and then challenged with KA 7 days later. Our results indicate that protein synthesis is necessary for protection in the piriform cortex and hippocampus. The need for protein synthesis in hippocampal protection agrees with other studies. Gage and Stanton w5x found that hypoxia preconditioning of rat hippocampal slices was blocked by either cycloheximide or actinomycin D Žan inhibitor of RNA synthesis.. The activation of hypoxia inducible factor-1 ŽHIF-1., a transcription factor induced by physiologically relevant O 2 reductions w6x, and respon-
sible for upregulation of hypoxia-associated genes was also prevented by these two agents w3x. Further evidence for transcription and translation Žstress response. involvement in preconditioning is given by the correlation of induction of HSP-70 to ischemia tolerance w12x. Also, enhanced synthesis of MnSOD occurs in hippocampus following preconditioning w7x. Thus, if stress proteinŽs. are induced by a specific stress, they may confer a generalized protection against a second challenge such as free radical generation. Synaptic adenosine release and receptor activation are also implicated in hypoxia-preconditioning protection against brain injury w4x. Adenosine and its receptors are abundant in several brain regions Ži.e., striatum, hippocampus, and cerebral cortex. w17x. Activation of adenosine receptor subtypes modulates excitatory amino acid transmission, ŽA1 s inhibition; A2 s stimulation; A3 s desensitization of the A1 response. w4x. Functioning in this role, adenosine would be neuroprotective in ischemia w16x. Adenosine may also function as an endogenous anticonvulsant w20x. Adenosine is released during hypoxia w14x, and may play a protective role by regulation of glutamate and Zn release. In fact, this may be why when rats exposed to an 8.5% O 2 , 91.5% N2 atmosphere 30 min after KA injection, seizures and hippocampal damage fail to develop w1x. In summary, hypoxia preconditioning provides neuroprotection against the development of KA-induced brain edema in areas sensitive to seizure-induced damage. This protective effect occurs 1 day following hypoxia and persists for 7 days. By 14 days, however, the neuroprotection is absent. These results indicate that hypoxia induces a general adaptive response that protects against the seizureinduced pathophysiology and may involve stress-related transcription factors and effector proteins.
Acknowledgements Supported in part by: DAAH04-95-0217. The authors express appreciation to Drs. Dennis Wallace and Jinshi Zhou for their statistical consultation.
References w1x S. Amano, T. Obata, F. Hazama, N. Kashiro, M. Shimada, Hypoxia prevents seizures and neuronal damages of the hippocampus induced by kainic acid in rats, Brain Res. 523 Ž1990. 121–126. w2x Y. Ben-Ari, Limbic seizure and brain damage produced by kainic acid: mechanisms and relevance to human temporal lobe epilepsy, Neuroscience 14 Ž1985. 375–403. w3x H.F. Bunn, R.O. Poyton, Oxygen sensing and molecular adaptation to hypoxia, Physiol. Rev. 76 Ž1996. 839–885. w4x T.V. Dunwiddie, L. Diao, H.O. Kim, J.L. Jiang, K.A. Jacobson,
M.R. Emerson et al.r Brain Research 825 (1999) 189–193
w5x
w6x w7x
w8x
w9x
w10x w11x
w12x
Activation of hippocampal adenosine A3 receptors produces a desensitization of A1 receptor-mediated responses in rat hippocampus, J. Neurosci. 17 Ž1997. 607–614. A.T. Gage, P.K. Stanton, Hypoxia triggers neuroprotective alterations in hippocampal gene expression via a heme-containing sensor, Brain Res. 719 Ž1996. 172–178. M. Gassmann, R.H. Wenger, HIF-1, a mediator of the molecular response to hypoxia, News Physiol. Sci. 12 Ž1997. 214–218. H. Kato, K. Kogure, T. Araki, X.H. Liu, K. Kato, Y. Itoyama, Immunohistochemical localization of superoxide dismutase in the hippocampus following ischemia in a gerbil model of ischemic tolerance, J. Cereb. Blood Flow Metab. 15 Ž1995. 60–70. K. Kitagawa, M. Matsumoto, M. Tagaya, R. Hata, H. Ueda, M. Niinobe, N. Handa, R. Fukunaga, K. Kimura, K. Mikoshiba et al., Ischemic tolerance phenomenon found in the brain, Brain Res. 528 Ž1990. 21–24. A. Lehmann, H. Hagberg, I. Jacobson, A. Hamberger, Effects of status epilepticus on extracellular amino acids in the hippocampus, Brain Res. 359 Ž1985. 147–151. S.M. Massa, R.A. Swanson, F.R. Sharp, The stress gene response in brain, Cerebrovasc. Brain Metab. Rev. 8 Ž1996. 95–158. S.R. Nelson, M.L. Mantz, J.A. Maxwell, Use of specific gravity in the measurement of cerebral edema, J. Appl. Physiol. 30 Ž1971. 268–271. S. Nishi, W. Taki, Y. Uemura, T. Higashi, H. Kikuchi, H. Kudoh,
w13x
w14x w15x w16x
w17x
w18x w19x
w20x
193
M. Satoh, K. Nagata, Ischemic tolerance due to the induction of HSP70 in a rat ischemic recirculation model, Brain Res. 615 Ž1993. 281–288. T.L. Pazdernik, M. Layton, S.R. Nelson, F.E. Samson, The osmoticrcalcium stress theory of brain damage: are free radicals involved?, Neurochem. Res. 17 Ž1992. 11–21. W. Pohle, C. Rauca, Hypoxia protects against the neurotoxicity of kainic acid, Brain Res. 644 Ž1994. 297–304. S.M. Rothman, J.W. Olney, Glutamate and the pathophysiology of hypoxic–ischemic brain damage, Ann. Neurol. 19 Ž1986. 105–111. K.A. Rudolphi, P. Schubert, F.E. Parkinson, B.B. Fredholm, Neuroprotective role of adenosine in cerebral ischaemia, Trends Pharmacol. Sci. 13 Ž1992. 439–445. R.E. Simpson, M.H. O’Regan, L.M. Perkins, J.W. Phillis, Excitatory transmitter amino acid release from the ischemic rat cerebral cortex: effects of adenosine receptor agonists and antagonists, J. Neurochem. 58 Ž1992. 1683–1690. G. Sperk, Kainic acid seizures in the rat, Prog. Neurobiol. 42 Ž1994. 1–32. J.V. Wade, F.E. Samson, S.R. Nelson, T.L. Pazdernik, Changes in extracellular amino acids during soman- and kainic acid-induced seizures, J. Neurochem. 49 Ž1987. 645–650. D. Young, M. Dragunow, Status epilepticus may be caused by loss of adenosine anticonvulsant mechanisms, Neuroscience 58 Ž1994. 245–261.
Short communication
Hypoxia preconditioning attenuates brain edema associated with kainic acid-induced status epilepticus in rats Mitchell R. Emerson b
a,b
, Stanley R. Nelson b , Fred E. Samson b , Thomas L. Pazdernik
a,b,)
a Department of Pharmacology, Toxicology, and Therapeutics, UniÕersity of Kansas Medical Center, Kansas City, KS 66160-7417, USA Smith Mental Retardation and Human DeÕelopment Research Center, UniÕersity of Kansas Medical Center, Kansas City, KS 66160-7417, USA
Accepted 26 January 1999
Abstract Kainic acid ŽKA.-induced seizures elicit edema associated with necrosis in susceptible brain regions Že.g., piriform cortex and hippocampal CA 1 and CA 3 regions.. To test the hypothesis that hypoxia preconditioning protects against KA-induced edema formation, adult male rats were exposed to a 9% O 2 , 91% N2 atmosphere for 8 h. KA Ž14 mgrkg, i.p.. was administered 1, 3, 7, or 14 days later. Regional analysis of edema indicated that hypoxia exposure attenuated edema formation in piriform and frontal cortices and hippocampus when KA was given 1, 3, or 7 days later but not 14 days after hypoxia. Cycloheximide Ž2 mgrkg s.c.. given 1 h prior to hypoxia prevented the protective effect of hypoxia on KA-induced edema attenuation in the piriform cortex and hippocampus. Thus, hypoxic challenge induces a general adaptive response that protects against the seizure-associated pathophysiology, with no direct relationship to seizure intensity. This response may involve stress-related transcription factors and effector proteins. q 1999 Elsevier Science B.V. All rights reserved. Keywords: Hypoxia preconditioning; Kainic acid; Status epilepticus; Seizure; Brain edema; Specific gravity
Hypoxiarischemia preconditioning paradigms can produce neuroprotection against subsequent brain insults. This phenomenon is based on the premise that a sublethal hypoxicrischemic episode will afford neuroprotection against later challenges even of a different kind w8x. The biochemical changes responsible for the preconditioning response are purported to include the stress-related proteins Žheat shock proteins w12x, superoxide dismutases w7x., as well as adenosine release andror receptor activation w4x. Systemic injection of kainic acid ŽKA. into rats causes a glutamate-driven status epilepticus that leads to a cellular calcium influx and oxidative stress similar to that seen in ischemia-reperfusion injury w13,15x. An early cytotoxic edema and a late vasogenic edema associated with necrosis occur in brain regions Ži.e., piriform cortex and hippocampal CA 1 and CA 3 regions. extensively involved in seizure activity w2,18x. KA augments the release of glutamate from neurons located in the hippocampus and piriform cortex w9,19x. Excess stimulation of glutamatergic pathways increases intracellular Naq and Ca2q concentrations leading
)
Corresponding [email protected]
author.
Fax:
q 1-913-588-5677;
E-mail:
to cytotoxic osmotic swelling of neurons and glia. As neuronal damage progresses to necrosis, a vasogenic edema results from reduced integrity of vessel endothelial walls and collapse of the blood–brain barrier w18x. Pohle and Rauca w14x report that exposure to mild hypoxia decreases the incidence of KA-induced seizures and thus lethality, as well as the KA-associated neuropathology when challenge is given 1 week later. The purpose of our study was to examine the effects of hypoxia preconditioning on KA-induced seizures and subsequent brain edema. The extent of protection as a function of the interval between hypoxia and KA challenge and the requirement for protein synthesis was studied. Adult male Wistar rats Ž250–350 g; Harlan Sprague– Dawley, Indianapolis, IN. were kept under standard conditions including a 12-h lightrdark cycle with access to food and water ad libitum. Rats were exposed to either a mild hypoxic Ž9% O 2 , 91% N2 . atmosphere ŽPuritan-Bennett, Kansas City, KS. in a flow-through chamber or to normal laboratory air for 8 h during the dark cycle. At timepoints of 1, 3, 7, or 14 days later, rats were injected with 0.9% saline as control or KA Ž14 mgrkg i.p.. dissolved in 0.9% saline. Twenty-four hours after KA or saline administration, rats were anesthetized with halothane, decapitated
0006-8993r99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved. PII: S 0 0 0 6 - 8 9 9 3 Ž 9 9 . 0 1 1 9 5 - 6
190
M.R. Emerson et al.r Brain Research 825 (1999) 189–193
and brains were removed for specific gravity measurements. For the protein synthesis studies, cycloheximide Ž2 mgrkg s.c.. dissolved in 0.9% saline was given 1 h prior to the 8 h of hypoxia or laboratory air exposure. Immediately after removal, brains were dissected under cold Ž48C. kerosene. Specific gravities were determined on samples about 2 mm3 in volume from brain regions of interest using a density gradient column consisting of kerosene and bromobenzene w11x. In edematous brain re-
gions, a decrease in specific gravity of brain tissue occurs as a result of the influx of fluid w11x. The percent tissue volume change was calculated from the specific gravity by the equation: % change in tissue volume as water s
Ž sp. gr.y 1 . cont.r Ž sp. gr.y 1 . exp. 4 y 1 = 100
where: sp. gr.s specific gravity; cont.s control; and exp.s experimental.
Fig. 1. Temporal effect of hypoxia preconditioning on KA-induced edema Žspecific gravity reduction. in various brain regions. ŽA. Hypoxia exposure prevented the formation of edema in the piriform cortex when KA challenge was given 1, 3, or 7 days later. Increases in tissue volume due to influx of water were detected following KA alone Ž9.58 " 1.71%. and KA-14 days post-hypoxia Ž24.54 " 2.02%.. ŽB. Hypoxia exposure prevented the formation of edema in the frontal cortex when KA challenge was given 1, 3, or 7 days later. Increases in tissue volume due to influx of water were detected following KA alone Ž6.27 " 2.10%. and KA-14 days post-hypoxia Ž18.45 " 8.50%.. ŽC. Hypoxia exposure prevented the formation of edema in the hippocampus when KA challenge was given 1, 3, or 7 days later. Increases in tissue volume due to influx of water were detected following KA alone Ž2.23 " 0.92%. and KA-14 days post-hypoxia Ž5.24 " 2.65%.. ŽD. The cerebellar vermis is considered non-responsive to KA, however, a decrease in tissue volume in the KA-1 day post-hypoxia group Žy4.65 " 1.85%. and an increase in the KA-7 days post-hypoxia group Ž1.78 " 1.13%. were detected possibly representing the heterogeneity of sampling in this tissue. All columns and bars represent the mean" S.E.M. Saline Ž n s 12.; KA Ž n s 29.; 1 day Ž n s 5.; 3 days Ž n s 10.; 7 days Ž n s 11.; and 14 days Ž n s 4.. U P - 0.05, significantly different from KA ŽKruskal–Wallis..
M.R. Emerson et al.r Brain Research 825 (1999) 189–193
Statistical significance was determined using a nonparametric analysis of variance procedure ŽKruskal–Wallis.. This procedure was used since our graphical analysis did not provide evidence that our data was distributed normally with constant variance. Immediately following hypoxia, animals appeared in good condition with the exception of hyperventilation and less exploratory activity than rats kept outside of the hypoxia chamber. These behaviors ceased within minutes of removal from the chamber. Rats exposed to hypoxia lost almost 4 g of weight, indicating a mild stress. Non-exposed rats gained approximately 9 g, a normal weight gain during the dark cycle. Hypoxia-exposed rats regained lost
191
weight within 24–48 h and continued weight gain at a normal rate when compared to non-exposed rats Ždata not shown.. Twenty-four hours after KA-administration, rats lost approximately 10% of their body weight. There was no difference in weight loss following KA in rats that were exposed to hypoxia, and those that were not Ždata not shown.. After KA challenge, there was a decrease in the specific gravity values Ži.e., increased edema. in piriform and frontal cortices, and hippocampus, but not cerebellum. In the three affected regions, the specific gravity decreases were considerably less in animals exposed to hypoxia and injected with KA 1, 3, or 7 days later. On the other hand,
Fig. 2. Effect of CHX given 1 h prior to hypoxia preconditioning on KA-induced edema Žspecific gravity reduction. in various brain regions when KA challenge is given 7 days later ŽCHX-Hypox. vs. hypoxia preconditioning alone when KA challenge is given 7 days later ŽHypox.. ŽA. CHX reversed hypoxia’s attenuation of edema formation in the piriform cortex. ŽB. CHX had no effect on hypoxia’s attenuation of edema formation in the frontal cortex. ŽC. CHX reversed hypoxia’s attenuation of edema formation in the hippocampus. ŽD. CHX caused an increase in edema formation in the cerebellar vermis. All columns and bars represent the mean " S.E.M. Saline Ž n s 9.; KA Ž n s 29.; Hypox Ž n s 11.; CHX-Hypox Ž n s 8.. U P - 0.05, significantly different from Hypox ŽKruskal–Wallis..
192
M.R. Emerson et al.r Brain Research 825 (1999) 189–193
animals challenged with KA 14 days following hypoxia exposure were not protected against the development of edema in the brain regions studied ŽFig. 1.. Cycloheximide given 1 h prior to hypoxia exposure significantly reversed the protection in the hippocampus and piriform cortex when the KA challenge was given 7 days later. The response in the frontal cortex was not significantly altered by cycloheximide pretreatment ŽFig. 2.. Hypoxia preconditioning protected against brain damage from KA-induced seizures assessed by specific gravity measurements of edema in susceptible brain regions. Hypoxia produced a mild stress as evident by hyperventilation, decreased exploratory behavioral activity and a modest weight loss. This mild stress initiates neurochemical changes, some of which require protein synthesis, that confer neuroprotection when rats are given a subsequent challenge such as KA-induced seizures. Pohle and Rauca w14x previously reported that exposure to mild hypoxia for 8 h provides neuroprotection against KA-induced histopathologic damage when the challenge was given 7 days later, primarily through reduction in seizure intensity. In our studies, the protection against edema formation was relatively short-lived Ži.e., 1 day through 7 days, absent at 14 days. and required protein synthesis in the limbic brain regions. However, there was no difference among groups in seizure intensity when lethality was included in the scoring system. Thus, it is notable that protection did occur in animals with intense but not lethal seizures. An adaptive response may be a contributing factor in protection conferred by hypoxia preconditioning. The stress response includes an alteration in gene expression induced by adverse conditions, such as ischemia or excitotoxicity w10x. Stress-related genes are activated under a variety of conditions and their effector proteins may participate in the protective response to KA-induced seizures. Some of these include the heat shock proteins, superoxide dismutases, metallothioneins, and heme oxygenases w7,10,12x. Which genes are altered during a specific response will depend on the nature and severity of the insult Ži.e., which transcription factors are inducedrrepressed during and after the insult.. To examine the dependence on protein synthesis in the neuroprotective effect, Ži.e., gene expression of protective proteins. rats were pretreated with cycloheximide 1 h prior to hypoxia exposure, and then challenged with KA 7 days later. Our results indicate that protein synthesis is necessary for protection in the piriform cortex and hippocampus. The need for protein synthesis in hippocampal protection agrees with other studies. Gage and Stanton w5x found that hypoxia preconditioning of rat hippocampal slices was blocked by either cycloheximide or actinomycin D Žan inhibitor of RNA synthesis.. The activation of hypoxia inducible factor-1 ŽHIF-1., a transcription factor induced by physiologically relevant O 2 reductions w6x, and respon-
sible for upregulation of hypoxia-associated genes was also prevented by these two agents w3x. Further evidence for transcription and translation Žstress response. involvement in preconditioning is given by the correlation of induction of HSP-70 to ischemia tolerance w12x. Also, enhanced synthesis of MnSOD occurs in hippocampus following preconditioning w7x. Thus, if stress proteinŽs. are induced by a specific stress, they may confer a generalized protection against a second challenge such as free radical generation. Synaptic adenosine release and receptor activation are also implicated in hypoxia-preconditioning protection against brain injury w4x. Adenosine and its receptors are abundant in several brain regions Ži.e., striatum, hippocampus, and cerebral cortex. w17x. Activation of adenosine receptor subtypes modulates excitatory amino acid transmission, ŽA1 s inhibition; A2 s stimulation; A3 s desensitization of the A1 response. w4x. Functioning in this role, adenosine would be neuroprotective in ischemia w16x. Adenosine may also function as an endogenous anticonvulsant w20x. Adenosine is released during hypoxia w14x, and may play a protective role by regulation of glutamate and Zn release. In fact, this may be why when rats exposed to an 8.5% O 2 , 91.5% N2 atmosphere 30 min after KA injection, seizures and hippocampal damage fail to develop w1x. In summary, hypoxia preconditioning provides neuroprotection against the development of KA-induced brain edema in areas sensitive to seizure-induced damage. This protective effect occurs 1 day following hypoxia and persists for 7 days. By 14 days, however, the neuroprotection is absent. These results indicate that hypoxia induces a general adaptive response that protects against the seizureinduced pathophysiology and may involve stress-related transcription factors and effector proteins.
Acknowledgements Supported in part by: DAAH04-95-0217. The authors express appreciation to Drs. Dennis Wallace and Jinshi Zhou for their statistical consultation.
References w1x S. Amano, T. Obata, F. Hazama, N. Kashiro, M. Shimada, Hypoxia prevents seizures and neuronal damages of the hippocampus induced by kainic acid in rats, Brain Res. 523 Ž1990. 121–126. w2x Y. Ben-Ari, Limbic seizure and brain damage produced by kainic acid: mechanisms and relevance to human temporal lobe epilepsy, Neuroscience 14 Ž1985. 375–403. w3x H.F. Bunn, R.O. Poyton, Oxygen sensing and molecular adaptation to hypoxia, Physiol. Rev. 76 Ž1996. 839–885. w4x T.V. Dunwiddie, L. Diao, H.O. Kim, J.L. Jiang, K.A. Jacobson,
M.R. Emerson et al.r Brain Research 825 (1999) 189–193
w5x
w6x w7x
w8x
w9x
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