Midkine, heparin-binding growth factor, blocks kainic acid-induced seizure and neuronal cell death in mouse hippocampus
- Yun B Kim†1, 2,
- Jae K Ryu†1,
- Hong J Lee1, 3,
- In J Lim4,
- Dongsun Park2,
- Min C Lee1, 5 and
- Seung U Kim1, 3Email author
© Kim et al; licensee BioMed Central Ltd. 2010
Received: 31 August 2009
Accepted: 26 March 2010
Published: 26 March 2010
Midkine (MK), a member of the heparin-binding growth factor family, which includes MK and pleiotrophin, is known to possess neurotrophic and neuroprotective properties in the central nervous system. Previous studies have shown that MK is an effective neuroprotective agent in reducing retinal degeneration caused by excessive light and decreasing hippocampal neuronal death in ischemic gerbil brain. The present study was undertaken to investigate whether MK acts as an anticonvulsant in kainic acid (KA)-induced seizure in mouse and blocks KA-mediated neuronal cell death in hippocampus.
Increased expression of MK was found in hippocampus of mouse following seizures induced by intracerebroventricular injection of KA, and MK expression was found in glial fibrillary acidic protein (GFAP)-positive astrocytes. Concurrent injection of MK and KA attenuated KA-induced seizure activity and cell death of hippocampal neurons including pyramidal cells and glutamic acid decarboxylase 67 (GAD67)-positive GABAergic interneurons in the CA3 and hilar area.
The results of the present study indicate that MK functions as an anticonvulsant and neuroprotective agent in hippocampus during KA-induced seizures.
Temporal lobe epilepsy (TLE) is pathologically characterized by extensive neuronal loss in the CA1, CA3 and hilar regions of hippocampus [1, 2]. Previous studies have demonstrated that the animal models of TLE generated by intracerebroventricular injection of kainic acid (KA) faithfully reproduce clinical and pathological features found in human TLE [3–7].
Previous studies have reported the possible involvement of neurotrophic factors in epilepsy as suggested by the gene expression of neurotrophic factors such as NGF, BDNF and NT-3 in hippocampus in human TLE as well as in TLE animal models [8, 9]. Midkine (MK), one of such neurotrophic factors, has emerged as an important neuromodulator in the central nervous system (CNS). MK, a member of the heparin-binding growth factor family, which includes MK and pleiotrophin, is known to possess neurotrophic and neuroprotective properties [10, 11]. MK was originally isolated as the product of retinoic acid-responsive gene that functions primarily in inducing cell differentiation in mouse teratocarcinoma cells , and has the ability to influence a variety of neuronal functions including neurite extension , neuronal differentiation [14, 15] and neuronal survival following injury or damage in the CNS [15, 16]. During the fetal development of the CNS, MK expression was demonstrated in neuroepithelial/neural progenitor cells following ethylnitrosourea injury  indicating that MK might have a role in cellular proliferation . Recent studies further showed that MK has been implicated in neurological diseases, including Alzheimer's disease , cerebral ischemia  and Parkinson-dementia complex of Guam (Lytico-bodig disease) . In patients with Alzheimer's disease  or Lytico-bodig disease , MK immunoreactivity was found in senile plaques and neurofibrillary tangles. In addition, an increased expression of MK was found in astrocytes in rat models of cerebral ischemia . It is not known, however, whether the expression of MK in the brain after the brain injury is a part of an endogenous repair process to prevent further damage in the CNS.
The objectives of the present study are to determine whether intracerebroventricularly injected MK acts as an anticonvulsant and blocks KA-mediated neuronal cell death in hippocampus.
MK expression after seizures
Interestingly, we have found the number of MK-positive cells markedly increased in stratum lacunosum moleculare (Slm) area of CA3 at 24-hr post-KA injection relative to PBS controls. Representative MK immunostaining in CA3 Slm is shown in Figure 1B (box denotes area of high magnification, top panel). In PBS-injected brain, only small number of MK-positive cells was observed (Figure 1B, middle panel). Following injection of KA, CA3 Slm area demonstrated an increase in the number of MK-positive cells (Figure 1B, bottom panel). Double-labeling immunofluorescence staining was then used to investigate MK expression in glial cells in KA-injected hippocampus. Representative double staining for MK (green staining, Figure 1C top panel) and GFAP (red staining, Figure 1C middle panel) showed that MK was expressed in GFAP-positive astrocytes; there was no MK expression in anti-complement receptor 3 (OX-42)-positive microglia (data not shown).
Anticonvulsant effect of MK
Effects of MK on KA-induced seizure activities
Latency to onset
(maximum score 5.0)
0.0 ± 0.0
0.0 ± 0.0
0.0 ± 0.0
KA (0.2) alone
169.9 ± 38.8*
1,200.6 ± 372.3*
4.2 ± 0.1*
160.0 ± 28.5
616.7 ± 50.3#
2.5 ± 0.3#
172.8 ± 33.8
422.4 ± 71.5#
2.2 ± 0.2#
188.8 ± 47.0
318.6 ± 31.7#
1.7 ± 0.3#
Neuroprotective effect of MK
The main finding of this study is that intracerebroventricular administration of MK conferred neuroprotection against KA-induced excitotoxic cell death of hippocampal neurons. Our results also demonstrated that MK was effective in attenuating KA-induced seizures and degeneration of GABAergic interneurons in hippocampus. The results from immunohistochemical staining showed that damaged pyramidal neurons in hippocampus are correlated with the decreased level of MK expression (Figure 1A). At present, it is not known whether this reduced level of MK immunoreactivity in neurons is causative factor for neuronal degeneration induced by KA injection. However, previous in vivo studies have demonstrated that MK is an effective neuroprotective agent in reducing retinal degeneration caused by excessive light  and decreasing hippocampal neuronal death in ischemic gerbil brain . Furthermore, MK knock-out mice displayed altered expression of calcium-binding protein in hippocampus and defective working memory . In view of these observations, it is possible that MK is involved in the regulation of endogenous neuroprotective process against externally applied injury or disease.
We have found that the level of MK immunoreactivity markedly increased in astrocytes after KA injection (Figure 1C). In a previous report, we have demonstrated rapid activation of astrocytes in hippocampal alveus and fimbria, strata oriens and lacunosum moleculare as well as dentate hilus as early as 1 hr following diisopropyl fluorophosphate (DFP)-induced seizures and neuronal injury . These observations suggest that the activated astrocytes serve as an important source of MK in response to excitotoxicity. Important findings in the present study are therapeutic and neuroprotective effects of MK on KA-induced neuronal injury in CA3 and dentate hilus of hippocampus (Figure 3). Our results are in good agreement with a previous study of neuroprotective effect of MK in transient forebrain ischemia when given immediately before middle cerebral artery occlusion . Previous studies have demonstrated that KA-injected brain show dramatic reduction in the number of GAD-positive interneurons [7, 23, 28], even in CA1 statum oriens and alveus where pyramidal neurons are relatively spared [Figures 3-5], which can cause abnormal functional inhibition of neuronal circuitry leading to hippocampal hyperexcitability [2, 7]. Thus, it is noteworthy that icv application of MK blocks the degeneration of GAD67-positive interneurons, especially in the stratum pyramidale and radiatum of CA3, and the molecular layer, granule cell layer and hilus of dentate gyrus as caused by KA injection (Figures 4 and 5).
The majority of the animals experienced stage 4/5 seizure severity after KA injection. A previous study has demonstrated in KA-injected rat epilepsy model that dizocilpine (MK-801) inhibited hippocampal neuronal loss without blocking seizure development . In addition, several studies in animal model of TLE have also shown that significant neuronal loss is not necessarily a prerequisite for the development of seizures [31, 32]. Such results imply that the mechanism of MK action in TLE model is different from that of MK-801, an NMDA antagonist. Our results show that MK-induced neuroprotection against KA toxicity is primarily associated with moderation of seizure activity (Table 1). It is believed that MK plays a role as an anticonvulsant directly (Table 1) or indirectly by preserving an inhibitory amino acid (GABA) system including GAD67-positive interneurons (Figures 4 and 5). Furthermore, it is possible to speculate that MK could be a neurotrophic factor, especially for GAD67-positive interneurons or astrocytes expressing high levels of MK (Figure 1) which may contribute to early cessation of seizure and control of its recurrence.
The exact mechanism of the neuroprotective activity of MK remains to be further clarified but activation of gene product(s) associated with apoptosis by MK may provide some answers. For instance, previous studies have shown that MK inhibits apoptotic process by up-regulation of bcl-2 expression  and by inhibition of caspase-3 activation . Thus, it is possible that the MK-mediated neuroprotective mechanism involves activation of signal transduction pathways involved in regulation of apoptotic cell death.
The results of the present study demonstrate that the administration of MK produces a significant neuroprotective effect against KA-induced neuronal loss in mouse model of epilepsy. Additional studies of MK to elucidate their neuroprotective activity in animal models of brain injury and neurodegeneration such as Parkinson's disease, Huntington's disease, amyotropic lateral sclerosis, stroke or spinal cord injury should prove MK as a member of neurotrophic factors that are valuable in providing effective treatment for patients with various neurological disorders.
Treatment and seizure monitoring
Male C57BL/6 mice (n = 8/group) weighing 25 - 30 g were used for the experiments. The animals were housed in a temperature- and humidity-controlled room that was kept on an alternating 2-hr light/dark schedule. Food and water were available ad libitum throughout the experiments. All animal experiments were conducted in accordance with the on Animal Care Committee of the University of British Columbia.
Animals were anesthetized with intraperitoneal injection of chloral hydrate (7%, 0.1 mL/kg) and then mounted in a stereotaxic apparatus (David Kopf Instruments, Tujunga, CA). KA (0.2 μg/0.4 μL PBS/mouse; Sigma, St. Louis, MO) was injected into the right lateral cerebroventricle at the coordinate (AP, -2.0; ML, -2.9; DV, -3.8) using a 10 μL Hamilton syringe fitted with 26G needle at a rate of 0.1 μL/min. The needle was left in place for 5 min. MK (0.1, 0.2 or 0.4 μg/0.4 μL PBS; LG Biotech, Daejon, Korea) was unilaterally co-injected with KA into the right lateral cerebroventricle. For sham-operated animals, the same volume of 0.1 M PBS was stereotaxically injected into the same coordinates described above. Wounds were sutured, and animals were allowed to recover and then returned to their cages. After KA injection, each animal was placed in a Plexiglas cylinder and their seizure behaviors - latency to onset, duration, and intensity - were recorded for a period of 120 min in a blind manner. Seizure intensity was scored with a slight modification from a previous scoring system  as followed: stage 1, immobilization and staring; stage 2, head nodding; stage 3, rearing accompanied by forelimb clonus and wet dog shakes; stage 4, falling and wobbling; stage 5, jumping, circling, or rolling. The starting time of head nodding was considered onset time of seizures, because the starting of immobilization and staring behaviors (stage 1) was not clear.
The animals were anesthetized and transcardially perfused with 50 mL cold saline followed by 100 mL of 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) 24 hr after KA administration. The brains were removed from the skull and post-fixed in 4% paraformaldehyde for 24 hr, followed with cryoprotection in 30% sucrose in phosphate buffer for 2 days. Serial coronal sections at 30 μm were prepared on a cryostat (CM 1900; Leica, Heerbrugg, Switzerland). Free-floating sections were prepared from the brains of PBS, KA, and KA plus MK-injected mice. For single immunofluorescence staining, brain sections were incubated in PBS containing 5% normal goat serum and 0.2% Triton X-100 for 30 min at room temperature (RT), and then incubated overnight with rabbit anti-MK antibody (1:500, kindly provided by Dr. T. Muramatsu, Nagoya University, Japan). Sections were then incubated with Alexa Fluor 488-conjugated goat anti-rabbit IgG (1:200; Molecular Probes, Eugene, OR) at RT for 2 hr in the dark. Sections were washed in phosphate buffer, mounted on slides. Nissl staining was also performed on slide-mounted brain sections with 0.1% cresyl violet (Sigma) for the evaluation of hippocampal neuronal loss.
For GAD67 immunohistochemical staining, all incubation solutions did not contain Triton X-100. The brain sections were briefly quenched with 3% H2O2 in PBS for 10 min and incubated with 5% normal goat serum for 30 min. The sections were incubated overnight at 4°C with rabbit anti-GAD67 (1:1000; Chemicon, Temecula, CA). The sections were then incubated for 1 hr with biotinylated anti-rabbit IgG (1:200; Vector, Burlingame, CA), followed by incubation with avidin-biotin complex (1:200, Vector) for 1 hr and then visualized with 0.05% 3,3'-diaminobenzidine (Sigma) and 0.003% H2O2. Negative control sections were prepared for immunohistochemical staining in an identical manner except the primary antibodies were omitted.
Double-labeling immunofluorescence microscopy
Free floating sections were incubated in PBS containing 3% normal goat serum and 0.3% Triton X-100 for 30 min at room temperature (RT). Brain sections were incubated for 48 hr at 4°C in a mixture of two primary antibodies: MK (1:100) in combination with mouse anti-GFAP (1:500; Sigma) or mouse OX-42 (1:200; Serotec, Oxford, UK). Sections were then incubated in a mixture of Alexa Fluor 488-conjugated goat anti-rabbit IgG (1:200; Molecular Probes) and Alexa Fluor 594-conjugated goat anti-mouse IgG (1:200; Molecular Probes) at RT for 2 hr in the dark. Processed sections were mounted on gelatin-coated slides, coversliped and examined under a Zeiss Axioplan-2 microscope.
Five coronal hippocampal sections (at the level of the injection site and spaced 60 μm from each other) were used for immunohistochemical analysis. All quantitative analyses were performed in a blind manner. To ensure consistency in tissue sampling, matched hippocampal sections (based on anatomical landmark) were always processed throughout the experiments. Digitized images of stained sections were acquired using a Zeiss Axioplan-2 microscope equipped with a DVC camera (Diagnostic Instruments, Sterling Heights, MI). These images were then analyzed using Northern Eclipse software (Empix Imaging, Mississauga, ON, Canada). Three microscopic fields within SP (stratum pyramidale) and Slm (stratum lacunosum moleculare) areas in CA3 were selected (magnification of ×40) in MK-stained sections. MK immunoreactivity was then measured and expressed as area density of MK (the fraction of the total given area occupied by MK-positive cells). Three microscopic fields (magnification at ×40) in CA1, CA3 and hilus region were counted for Nissl-stained undamaged neurons in each coronal brain section. Neurons are identified by dark nucleoli within lightly stained nuclei. GAD67-positive neurons were quantified in CA1 and CA3 subfields (strata oriens, radiatum, and pyramidale) and dentate gyrus layers (dentate hilus, granule cell layer, and molecular layer). These regions were established according to the atlas of Paxinos & Watson . Number of Nissl- and GAD67-positive neurons was expressed as a percentage of the PBS-injected control sections.
Data are presented as means SEM. The statistical significance was determined by one-way ANOVA and Student-Newman-Keuls test or Student's t-test using the StatView program (Abacus Concepts, Berkeley, CA). p values < 0.05 were considered to be statistically significant.
This work was supported by grants from the National R&D Program for Cancer Control, Korean Ministry of Health and Welfare and the Canadian Myelin Research Initiative.
- Bruton CJ: The neuropathology of temporal lobe epilepsy. 1988, New York, Oxford University PressGoogle Scholar
- Sloviter RS: The functional organization of the hippocampal dentate gyrus and its relevance to the pathogenesis of temporal lobe epilepsy. Ann Neurol. 1994, 35: 640-654. 10.1002/ana.410350604.View ArticlePubMedGoogle Scholar
- Nadler JV, Perry BW, Cotman CW: Intraventricular kainic acid preferentially destroys hippocampal pyramidal cells. Nature. 1978, 271: 676-677. 10.1038/271676a0.View ArticlePubMedGoogle Scholar
- Franck JE: Dynamic alterations in hippocampal morphology following intra-ventricular kainic acid. Acta Neuropathol. 1984, 62: 242-253. 10.1007/BF00691859.View ArticlePubMedGoogle Scholar
- Ben-Ari Y: Limbic seizures and brain damage produced by kainic acid: Mechanisms and relevance to human temporal lobe epilepsy. Neuroscience. 1985, 14: 375-403. 10.1016/0306-4522(85)90299-4.View ArticlePubMedGoogle Scholar
- Sperk G: Kainic acid seizures in the rat. Prog Neurobiol. 1994, 42: 1-32. 10.1016/0301-0082(94)90019-1.View ArticlePubMedGoogle Scholar
- Shetty AK, Turner DA: Fetal hippocampal grafts containing CA3 cells restore host hippocampal glutamate decarboxylase-positive interneuron numbers in a rat model of temporal lobe epilepsy. J Neurosci. 2000, 20: 8788-8801.PubMedGoogle Scholar
- Gall CM, Isackson PJ: Limbic seizures increase neuronal production of messenger RNA for nerve growth factor. Science. 1989, 245: 758-761. 10.1126/science.2549634.View ArticlePubMedGoogle Scholar
- Mathern GW, Babb TL, Micevych PE, Blanco CE, Pretorius JK: Granule cell mRNA levels for BDNF, NGF, and NT-3 correlate with neuron losses or supragranular mossy fiber sprouting in the chronically damaged and epileptic human hippocampus. Mol Chem Neuropathol. 1997, 30: 53-76. 10.1007/BF02815150.View ArticlePubMedGoogle Scholar
- Rauvala H: An 18-kd heparin-binding protein of developing brain that is distince tfrom fibroblast growth factors. EMBO J. 1989, 8: 2933-2941.PubMed CentralPubMedGoogle Scholar
- Muramatsu H, Shirahama H, Yonezawa S, Maruta H, Muramatsu T: Midkine, a retinoic acid-inducible growth/differentiation factor: immunochemical evidence for the function and distribution. Dev Biol. 1993, 159: 392-402. 10.1006/dbio.1993.1250.View ArticlePubMedGoogle Scholar
- Kadomatsu K, Tomomura M, Muramatsu T: cDNA cloning and sequencing of a new gene intensely expressed in early differentiation stages of embryonal carcinoma cells and in midgestation period of mouse embryogenesis. Biochem Biophys Res Commun. 1988, 151: 1312-1318. 10.1016/S0006-291X(88)80505-9.View ArticlePubMedGoogle Scholar
- Muramatsu H, Muramatsu T: Purification of recombinant midkine and examination of its biological activities: functional comparison of new heparin binding factors. Biochem Biophys Res Commun. 1991, 177: 652-658. 10.1016/0006-291X(91)91838-4.View ArticlePubMedGoogle Scholar
- Michikawa M, Kikuchi S, Muramatsu H, Muramatsu T, Kim SU: Retinoic acid responsive gene product, midkine, has neurotrphic functions in mouse spinal cord and dorsal root ganglion neurons in culture. J Neurosci Res. 1993, 35: 530-539.View ArticlePubMedGoogle Scholar
- Satoh J, Muramatsu H, Moretto G, Chang HJ, Kim ST, Cho JM, Kim SU: Midkine that promotes survival of fetal human neurons is produced by fetal astrocytes in culture. Brain Res Dev Brain Res. 1993, 75: 201-205. 10.1016/0165-3806(93)90024-5.View ArticlePubMedGoogle Scholar
- Kikuchi S, Muramatsu H, Muramatsu T, Kim SU: Midkine, a novel neurotrophic factor, promotes survival of mesencephalic neurons in culture. Neurosci Lett. 1993, 160: 9-12. 10.1016/0304-3940(93)90904-Y.View ArticlePubMedGoogle Scholar
- Kikuchi-Horie K, Kawakami E, Kamata M, Wada M, Hu JG, Ohara K, Watabe K, Oyanagi K: Distinctive expression of midkine in the repair period of rat brain during neurogenesis: immunohistochemical and immunoelectron microscopic observations. J Neurosci Res. 2004, 75: 678-687. 10.1002/jnr.20015.View ArticlePubMedGoogle Scholar
- Ratovitski EA, Kotzbauer PT, Milbrandt J, Lowenstein CJ, Burrow CR: Midkine induces tumor cell proliferation and binds to a high affinity signaling receptor associated with JAK tyrosine kinases. J Biol Chem. 1998, 273: 3654-3660. 10.1074/jbc.273.6.3654.View ArticlePubMedGoogle Scholar
- Yasuhara O, Muramatsu H, Kim SU, Muramatsu T, Maruta H, McGeer PL: Midkine, a novel neurotrophic factor, is present in senile plaques of Alzheimer disease. Brain Res Dev Brain Res. 1993, 192: 246-251.Google Scholar
- Yoshida Y, Goto M, Tsutsui J: Midkine is present in the early stage of cerebral infarct. Brain Res Dev Brain Res. 1995, 85: 25-30. 10.1016/0165-3806(94)00183-Z.View ArticlePubMedGoogle Scholar
- Yasuhara O, Schwab C, Matsuo A, Kim SU, Steele JC, Akiguchi I, Kimura H, McGeer EG, McGeer PL: Midkine-like immunoreactivity in extracellular neurofibrillary tangles in brains of patients with parkinsonism-dementia complex of Guam. Neurosci Lett. 1996, 205: 107-110. 10.1016/0304-3940(96)12385-5.View ArticlePubMedGoogle Scholar
- Mochizuki R, Takeda A, Sato N, Kimpara T, Onodera H, Itoyama Y, Muramatsu T: Induction of midkine expression in reactive astrocytes following rat transient forebrain ischemia. Exp Neurol. 1998, 149: 73-78. 10.1006/exnr.1997.6687.View ArticlePubMedGoogle Scholar
- Buckmaster PS, Jongen-Relo AL: Highly specific neuron loss preserves lateral inhibitory circuits in the dentate gyrus of kainate-induced epileptic rats. J Neurosci. 1999, 19: 9519-9529.PubMedGoogle Scholar
- Unoki K, Ohba N, Arimura H, Muramatsu H, Muramatsu T: Rescue of photoreceptors from the damaging effects of constant light by midkine, a retinoic acid-responsive gene product. Invest Ophthal Vis Sci. 1994, 35: 4063-4068.PubMedGoogle Scholar
- Yoshida Y, Ikematsu S, Moritoyo T, Goto M, Tsutsuji J, Sakuma S, Osame M, Muramatsu T: Intraventricular administration of the neurotrophic factor midkine ameliorates hippocampal delayed neuronal death following transient forebrain ischemia in gerbils. Brain Res. 2001, 894: 46-55. 10.1016/S0006-8993(00)03209-1.View ArticlePubMedGoogle Scholar
- Nakamura E, Kadomatsu K, Yuasa S, Muramatsu H, Nabeshima T, Fan QW, Ishiguro K, Igakura T, Matsubara S, Kaname T, Horiba M, Saaito H, Muramatsu T: Disruption of the midkine gene (Mdk) resulted in altered expression of a calcium binding protein in the hippocampus of infant mice and their abnormal behavior. Genes Cells. 1998, 12: 811-822. 10.1046/j.1365-2443.1998.00231.x.View ArticleGoogle Scholar
- Kim Y-B, Hur G-H: Seizure-related encephalopathy in rats intoxicated with diisopropylfluorophosphate. J Toxicol Pub Health. 2001, 17: 73-82.Google Scholar
- Houser CR, Esclapez M: Vulnerability and plasticity of the GABA system in the pilocarpine model of spontaneous recurrent seizures. Epilepsy Res. 1996, 26: 207-218. 10.1016/S0920-1211(96)00054-X.View ArticlePubMedGoogle Scholar
- Morin F, Beaulieu C, Lacaille J-C: Selective loss of GABA neurons in area CA1 of the rat hippocampus after intraventricular kainite. Epilepsy Res. 1998, 32: 363-369. 10.1016/S0920-1211(98)00033-3.View ArticlePubMedGoogle Scholar
- Brandt C, Potschka H, Löscher W, Ebert U: N-methyl-D-aspartate receptor blockade after status epilepticus protects against limbic brain damage but not against epilepsy in the kainate model of temporal lobe epilepsy. Neuroscience. 2003, 118: 727-740. 10.1016/S0306-4522(03)00027-7.View ArticlePubMedGoogle Scholar
- Bertram EH, Scott C: The pathological substrate of limbic epilepsy: neuronal loss in the medial dorsal thalamic nucleus as the consistent change. Epilepsia. 2000, 41 (Suppl 6): S3-S8. 10.1111/j.1528-1157.2000.tb01548.x.View ArticlePubMedGoogle Scholar
- Raol YS, Budreck EC, Brooks-Kayal AR: Epilepsy after early-life seizures can be independent of hippocampal injury. Ann Neurol. 2003, 53: 503-511. 10.1002/ana.10490.View ArticlePubMedGoogle Scholar
- Qi M, Ikematsu S, Ichihara-Tanaka K, Sakuma S, Muramatsu T, Kadomatsu K: Midkine rescues Wilms' tumor cells from cisplatin-induced apoptosis: regulation of Bcl-2 expression by midkine. J Biochem. 2000, 127: 269-277.View ArticlePubMedGoogle Scholar
- Owada K, Sanjo N, Kobayashi T, Mizusawa H, Muramatsu H, Muramatsu T, Michikawa M: Midkine inhibits caspase-dependent apoptosis via the activation of mitogen-activated protein kinase and phosphatidylinositol 3-kinase in cultured neurons. J Neurochem. 1999, 73: 2084-2092.PubMedGoogle Scholar
- Benkovic SA, O'Callaghan JP, Miller DB: Sensitive indicators of injury reveal hippocampal damage in C57BL/6J mice treated with kainic acid in the absence of tonic-clonic seizures. Brain Res. 2004, 1024: 59-76. 10.1016/j.brainres.2004.07.021.View ArticlePubMedGoogle Scholar
- Paxinos G, Watson C: The rat brain in stereotaxic coordinates. 2005, Elsevier Academic Press, 5Google Scholar