- Email: [email protected]
Transgenic mouse overexpressing the Akt reduced the volume of infarct area after middle cerebral artery occlusion
Neuroscience Letters 359 (2004) 159–162 www.elsevier.com/locate/neulet
Transgenic mouse overexpressing the Akt reduced the volume of infarct area after middle cerebral artery occlusion Norihiro Ohbaa,b, Sumiko Kiryu-Seoa, Mitsuyo Maedaa, Michinari Muraokab, Masamitsu Ishiib, Hiroshi Kiyamaa,* a
Department of Anatomy and Neurobiology, Graduate School of Medicine, Osaka City University, 1-4-3 Asahimachi, Abeno-ku, Osaka, 545-8585, Japan b Department of Plastic and Reconstructive Surgery, Graduate School of Medicine, Osaka City University, 1-4-3 Asahimachi, Abeno-ku, Osaka, 545-8585, Japan Received 3 December 2003; received in revised form 7 February 2004; accepted 9 February 2004
Abstract Transgenic mouse lines expressing the active form Akt gene under the control of the damage-induced neuronal endopeptidase (DINE) promoter were made from three different founder mice, and its neuroprotective potential against ischemic brain damage was investigated. Twenty-four hours after middle cerebral artery occlusion, two DINE-Akt-transgenic mouse lines displayed reductions of the infarcted area by 35% compared to the wild-type littermate. RT-PCR assays showed a high level of transgene in response to ischemic brain damage in these lines. These results suggest that the DINE promoter is a useful promoter, which responds to neuronal insults and that the Akt-induced neuroprotective effect against ischemic damage is potent in vivo. q 2004 Elsevier Ireland Ltd. All rights reserved. Keywords: Akt; Transgenic mouse; Damage-induced neuronal endopeptidase; Endothelin Converting Enzyme Like (ECEL); Ischemia; Neuronal protection
Among the therapeutic strategies for treating cerebral ischemia, neuroprotective therapy aims to improve the viability of neurons by enhancing cell survival-promoting mechanisms. In this respect, the phosphoinositide 3-kinase (PI3-K) mediated signaling pathway, which is one of the most potent survival signaling pathways, may be a promising target of neuroprotective therapy for the treatment of ischemic neuronal damages. Along this pathway, Akt (protein kinase B; PKB), which is a serine/threonine protein kinase and activated by PI3-K, could be a pivotal molecule in mediating the cell survival signal. Akt suppresses functions of several pro-apoptotic molecules such as Bad, p53 and the forkhead transcription factors [2,3, 11], and thereby promotes the neuroprotective mechanism [7,8,12]. To evaluate the consequence of Akt activation in rescuing ischemia-induced neuronal death, we generated transgenic mice expressing Akt in neuronal cells. In this mice Akt expression is controlled by the promoter of the damage-induced neuronal endopeptidase (DINE), which is a * Corresponding author. Tel.: þ81-6-66-45-3701; fax: þ 81-6-66-453702. E-mail address: [email protected] (H. Kiyama).
unique membrane-bound metallopeptidase. DINE was initially isolated by differential display polymerase chain reaction using rat normal and axotomized hypoglossal motoneurons [6]. The most characteristic properties of DINE are neuron-specific expression and a striking expression response to a wide range of neuronal insults including brain ischemia [5]. DINE expression after middle cerebral artery (MCA) occlusion is observed in neuronal cells located near the infarction core site and most of the expressing cells are assumed to project their axons into the damaged core region. Considering this characteristic expression pattern of DINE, we speculated that the synthesis of constitutively active Akt could be locally induced in those damaged neurons after MCA occlusion, and thus this transgenic mouse would provide a useful tool to investigate the role of Akt in ischemia-related brain damage in vivo. All experimental procedures were conducted in accordance with the standard guidelines for animal experiments of the Graduate School of Medicine, Osaka City University. Transgenic mice carrying the human active form Akt gene under the control of the DINE promoter were generated. Fig. 1 shows the inserted construct of genes. In order to examine the amount of expression of the transgene, we
0304-3940/03/$ - see front matter q 2004 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2004.02.029
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N. Ohba et al. / Neuroscience Letters 359 (2004) 159–162
Fig. 1. Schematic representation of the transgene construct.
inserted the green fluorescent protein (GFP) reporter gene driven by an internal ribosomal entry site (IRES) in the downstream part of the Akt gene. GFP expression from transgenic mice was intended to monitor the synthesis of extrinsic Akt. The transgenic and wild-type (WT) littermates were generated by crossing onto a C57BL/6 background. Three lines of transgenic mice from the heterozygotes of transgenic mice originating from three different founder mice were established, maintained and used for all experiments. These three lines of transgenic mice were designated as line A – C. Unilateral permanent MCA occlusion was performed in mice (postnatal days 60 –90) as described previously [10]. Briefly, animals were anesthetized with halothane in a mixture of 70% nitrous oxide and 30% oxygen. A vertical incision was made between the orbit and external auditory canal, and subtemporal craniotomy was performed. MCA from distal to the olfactory tract was permanently occluded by bipolar electrocoagulation and transected to avoid recanalization. Then animals were allowed to recover in a cage at 37 8C for 1 h. The body temperature was maintained at 37 ^ 0.5 8C using a heating lamp and blanket. As repeated blood withdrawal is incompatible with survival, a separate group of mice (n ¼ 6 each) was used for evaluation of physiological parameters. A PE-10 femoral arterial catheter was used to sample arterial blood gases before and after MCA occlusion. Blood glucose and hematocrit were also examined. None of the physiological parameters differed significantly between transgenic and WT mice either before or after MCA occlusion (Table 1). Mice were sacrificed by decapitation at 24 h following MCA occlusion, and the brain was rapidly removed and sliced into 1 mm thick coronal sections using a brain slicer (RBS-01, Aster industries, USA). These sections were immersed in 2% 2,3,5-triphenyltetrazolium chloride (TTC) dissolved in 0.1 M phosphate buffered saline (PBS) in the dark for 30 min at 37 8C. The sections were then transferred to buffered 10% formalin for fixation overnight. The fixed sections were scanned into an image analysis system (Mac
SCOPE, Mitani corporation, Japan), and infarcts were traced at each level. The contribution of the volume increase due to the ischemic edema was corrected according to Swanson et al. [9]. All values are expressed as means ^ SD. All of the volume comparisons between transgenic and WT groups were analyzed by analysis of variance and multiple comparison test. P values of , 0.05 were considered statistically significant. RT-PCR assays were used to quantify the transgene levels in transgenic mouse lines. Total RNA was obtained from either the operated or the normal upper half of cortex at 24 h following MCA occlusion (n ¼ 3 each). Total RNA (each 0.2 mg) was converted to cDNA with superscript reverse transcriptase (Gibco/BRL) and nucleotide oligo dT18. Aliquots from the reverse transcription reaction were used for PCR amplification using primer pairs ubiquitously expressing glyceraldehyde 3-phosphatase dehydrogenase (GAPDH) as a control. The specific primers for the mRNA of Akt and GFP were used and amplified by PCR. The reaction products were separated electrophoretically on 1% agarose gel and visualized by UV photography with added ethidium bromide stain. Quantification was performed using an image scanner (GT-8200UF, EPSON, Japan) and image analysis software (Photoshop 6.0, Adobe Systems Incorporated, USA). The density of each GFP band was normalized against that of GAPDH (GFP/GAPDH). For immunohistochemistry, mice were anesthetized with diethyl ether and rapidly perfused transcardially with 0.9% sodium chloride, followed by perfusion with 4% paraformaldehyde in 0.1 M phosphate buffer (PB) at 1 day. The brain was immediately removed, and postfixed with the same solution overnight at 4 8C. The brain was then cryoprotected by storing the tissue in 30% sucrose in PB routinely, frozen in powdered dry ice and stored at 2 80 8C. Frontal sections including the ischemic region were cut at a thickness of 20 mm with a cryostat. Floating sections were pretreated with 0.3% H2O2 in Tris-buffered saline (TBS) for 30 min, rinsed three times in TBS, and incubated with TBS including 0.1% Triton X-100 containing 3% bovine serum albumin (BSA) for 1 h at room temperature (RT) to block nonspecific binding. The sections were incubated with mouse monoclonal antibody against phosphorylated Akt for 24 h at 4 8C (1:500, Cell Signaling Technology, Beverly, MA, USA) in TBS including 0.1% Triton X-100 containing
Table 1 Physiological variables Transgenic mouse
Arterial pH PaO2 (mmHg) PaCO2 (mmHg) Blood glucose (mg/dl) Hematocrit (%)
Wild-type
Pre-operation
Post-operation
Pre-operation
Post-operation
7.35 ^ 0.03 162.0 ^ 20.0 44.8 ^ 2.8 134.7 ^ 22.2 48.8 ^ 2.5
7.36 ^ 0.03 155.2 ^ 22.1 40.6 ^ 2.8 148.0 ^ 21.2 46.1 ^ 2.0
7.32 ^ 0.03 166.5 ^ 9.7 39.7 ^ 7.4 161.0 ^ 14.0 46.5 ^ 6.2
7.34 ^ 0.02 163.8 ^ 7.0 35.1 ^ 7.0 157.0 ^ 11.4 43.6 ^ 5.0
Values are means ^ SD from six animals in each group.
N. Ohba et al. / Neuroscience Letters 359 (2004) 159–162
1% BSA. They were then incubated with biotinylated antigoat IgG (1:200) for 1 h at RT, followed by incubation in ABC complex (Vector Laboratories, Inc., Burlingame, CA) for 1 h at RT. Sections were visualized by 3,30 -diaminobenzidine tetrahydrochloride –H2O2 solution and mounted onto 3-aminopropyltryethoxysilane-coated slides. All the transgenic mice belonging to the three lines appeared behaviorally normal, with no abnormalities in eating or sleeping activity. There was one apparent phenotypic difference (curly hair) in one of the pedigrees of the transgenic mouse (line B). Gross observation of transgenic mice brains did not reveal any dramatic differences in size or structure compared with the brains of their WT littermates. At 24 h after MCA occlusion, infarction size was quantified from a series of TTC stained sections (Fig. 2a). The infarct volumes of the three experimental lines were measured as mentioned before (Fig. 2b). The WT mice had a large consistent infarction of the left neocortex (15.172 ^ 5.076 mm3, n ¼ 18). The infarct sizes of line B (9.461 ^ 6.491 mm3, 37.6% reduction, n ¼ 12) and line C (9.838 ^ 6.485 mm 3, 35.2%
Fig. 2. (a) Panel of coronal slices reconstructing the forebrain 24 h after MCA occlusion representative of transgenic mouse line B (upper panel) and a WT animal (lower panel). The slices have been stained with TTC to show the area of infarction (white). (b) Determination of ischemic brain damage on WT and transgenic mice. The bar graph shows the volume of infarction in each line subjected to permanent focal cerebral ischemia. The mean infarct volumes were as follows: WT mice, 15.172 ^ 5.076 mm3, n ¼ 18; line A, 15.060 ^ 4.902 mm3, n ¼ 5; line B, 9.461 ^ 6.491 mm3, n ¼ 12; line C, 9.838 ^ 6.485 mm3, n ¼ 7. Mean infarct volumes were obtained by the summation of lesion areas in TTC staining and multiplied by slice-toslice distance. These volumes were corrected for brain swelling as described by Swanson et al. [9]. All values are expressed as means ^ SD. All of the volume comparisons between transgenic and WT lines were analyzed by analysis of variance and multiple comparison test. P , 0:05 was considered to indicate statistical significance. *P , 0:02, **P , 0:05.
161
reduction, n ¼ 7) were significantly smaller than that of the WT littermates or line A (15.060 ^ 4.902 mm3, 0.7% reduction, n ¼ 5). Several mice in line A repeatedly demonstrated relatively similar infarction size to that of WT littermates. The difference in infarct volume between WT and the transgenic mice (lines B and C) was statistically significant, as determined by analysis of variance and multiple comparison test. We examined the changes in Akt mRNAs by RT-PCR. The conditions of RT-PCR were carefully selected to prevent signal saturation. Permanent MCA occlusion resulted in up-regulation of expression of Akt mRNAs in the operated cortex (Fig. 3a). The expression level of GFP mRNA, which is a marker of exogenous expression of Akt mRNA, in the cerebral cortex was also evaluated by RTPCR analysis (Fig. 3a). We have attempted to detect the direct fluorescence of GFP on the tissue sections, however the fluorescence was not strong enough to observe with a fluorescent microscope. The expression levels of GFP mRNA in the normal cortices of line C were very low or below the level of detection, although the expression level in line B was significant even in the normal condition. At 24 h after MCA occlusion, a substantial increase of GFP mRNA in the operated side of cortices was observed in lines B and C (Fig. 3a). Fig. 3b demonstrates the magnitude of change in the relative transcript level of the GFP. Quantification of the bands by densitometry revealed that the GFP transcript level in the operated cortex of line B was 1.7 fold and of line C was 11.2 fold higher than that of nonoperated cortex. We carried out immunohistochemistry with anti-phospho-Akt antibody to investigate the expression of
Fig. 3. (a) Expression levels of Akt and GFP mRNA in transgenic mouse lines and WT detected by RT-PCR in control and operated cortex at 24 h following MCA occlusion. C, control side; O, operated side. Equivalent amounts of GAPDH were amplified from all samples, verifying standardization of conditions. (b) Quantification of results from the RTPCR shown in (a). The density of each GFP band was normalized against that of GAPDH (GFP/GAPDH). (c) Photomicrographs showing immunohistochemical localization of phospho-Akt-positive cells in the peri-infarct cortex of line B (left) and WT (right) at 1 day. Bar, 50 mm.
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active Akt protein in the brain following MCA occlusion. Phospho-Akt immunoreactive cells were seen in the periinfarct cortex of transgenic mice at 1 day after MCA occlusion, however such immunoreactivity was not seen in the WT mice (Fig. 3c). The present study demonstrates that damage-induced expression of the active Akt in mouse brain provides marked protection against ischemic damage in a model of permanent focal cerebral ischemia. Our observation adds further in vivo evidence on the functional consequences of Akt to the recent studies demonstrating the neuroprotective effects of Akt [7,8,12]. In this study, two Akt transgenic mouse lines (B and C) showed a marked increase of GFP mRNA levels in the damaged brain. One of the striking observations in this study is that the reduction of infarct volume seems to correlate with the expression level of trans-genes (AktGFP). The level of Akt-GFP mRNAs in line A after MCA occlusion might not be sufficient to provide significant tissue protection. The reason why line A failed to respond to neuronal injury is obscure, but the expression levels of the transgene could be modulated by the chromosomal position effect in a particular transgenic mouse line as the integration occurs at random chromosomal sites [1,4]. In the present study we firstly used the DINE promoter for the expression of transgene. DINE is normally expressed in a group of neurons mainly in the hypothalamus, but not in the cerebral cortex. In the transgenic mice, the expression level of transgenic Akt and GFP in normal cortex was low, but markedly induced in response to ischemic damage, which is very similar to the expression pattern of endogenous DINE. This suggests that the DINE promoter is a useful promoter for expression of molecules, which are crucial to promote rescue or neurite elongation activities in vivo. In conclusion, the protective effects of Akt under the control of the damage-specific promoter DINE after ischemic injury would provide a novel and potent therapeutic intervention in brain injuries.
Acknowledgements This study was supported in part by grants from MHLW and MEXT of Japan, Japan Brain Foundation, Novartis Foundation, and Osaka Gas Group Welfare Foundation.
References [1] R. al-Shawi, J. Kinnaird, J. Burke, J.O. Bishop, Expression of a foreign gene in a line of transgenic mice is modulated by a chromosomal position effect, Mol. Cell. Biol. 10 (1990) 1192– 1198. [2] A. Brunet, A. Bonni, M.J. Zigmond, M.Z. Lin, P. Juo, L.S. Hu, M.J. Anderson, K.C. Arden, J. Blenis, M.E. Greenberg, Akt promotes cell survival by phosphorylating and inhibiting a Forkhead transcription factor, Cell 96 (1999) 857–868. [3] S.R. Datta, H. Dudek, X. Tao, S. Masters, H. Fu, Y. Gotoh, M.E. Greenberg, Akt phosphorylation of BAD couples survival signals to the cell-intrinsic death machinery, Cell 91 (1997) 231–241. [4] D. Garrick, S. Fiering, D.I. Martin, E. Whitelaw, Repeat-induced gene silencing in mammals, Nat. Genet. 18 (1998) 56–59. [5] R. Kato, S. Kiryu-Seo, H. Kiyama, Damage-induced neuronal endopeptidase (DINE/ECEL) expression is regulated by leukemia inhibitory factor and deprivation of nerve growth factor in rat sensory ganglia after nerve injury, J. Neurosci. 22 (2002) 9410–9418. [6] S. Kiryu-Seo, M. Sasaki, H. Yokohama, S. Nakagomi, T. Hirayama, S. Aoki, K. Wada, H. Kiyama, Damage-induced neuronal endopeptidase (DINE) is a unique metallopeptidase expressed in response to neuronal damage and activates superoxide scavengers, Proc. Natl. Acad. Sci. USA 97 (2000) 4345–4350. [7] N. Noshita, A. Lewen, T. Sugawara, P.H. Chan, Evidence of phosphorylation of Akt and neuronal survival after transient focal cerebral ischemia in mice, J. Cereb. Blood Flow Metab. 21 (2001) 1442–1450. [8] Y.B. Ouyang, Y. Tan, M. Comb, C.L. Liu, M.E. Martone, B.K. Siesjo, B.R. Hu, Survival- and death-promoting events after transient cerebral ischemia: phosphorylation of Akt, release of cytochrome C and activation of caspase-like proteases, J. Cereb. Blood Flow Metab. 19 (1999) 1126–1135. [9] R.A. Swanson, M.T. Morton, G. Tsao-Wu, R.A. Savalos, C. Davidson, F.R. Sharp, A semiautomated method for measuring brain infarct volume, J. Cereb. Blood Flow Metab. 10 (1990) 290–293. [10] F.A. Welsh, T. Sakamoto, A.E. McKee, R.E. Sims, Effect of lactacidosis on pyridine nucleotide stability during ischemia in mouse brain, J. Neurochem. 49 (1987) 846 –851. [11] A. Yamaguchi, M. Tamatani, H. Matsuzaki, K. Namikawa, H. Kiyama, M.P. Vitek, N. Mitsuda, M. Tohyama, Akt activation protects hippocampal neurons from apoptosis by inhibiting transcriptional activity of p53, J. Biol. Chem. 276 (2001) 5256–5264. [12] S. Yano, M. Morioka, K. Fukunaga, T. Kawano, T. Hara, Y. Kai, J. Hamada, E. Miyamoto, Y. Ushio, Activation of Akt/protein kinase B contributes to induction of ischemic tolerance in the CA1 subfield of gerbil hippocampus, J. Cereb. Blood Flow Metab. 21 (2001) 351 –360.
Transgenic mouse overexpressing the Akt reduced the volume of infarct area after middle cerebral artery occlusion Norihiro Ohbaa,b, Sumiko Kiryu-Seoa, Mitsuyo Maedaa, Michinari Muraokab, Masamitsu Ishiib, Hiroshi Kiyamaa,* a
Department of Anatomy and Neurobiology, Graduate School of Medicine, Osaka City University, 1-4-3 Asahimachi, Abeno-ku, Osaka, 545-8585, Japan b Department of Plastic and Reconstructive Surgery, Graduate School of Medicine, Osaka City University, 1-4-3 Asahimachi, Abeno-ku, Osaka, 545-8585, Japan Received 3 December 2003; received in revised form 7 February 2004; accepted 9 February 2004
Abstract Transgenic mouse lines expressing the active form Akt gene under the control of the damage-induced neuronal endopeptidase (DINE) promoter were made from three different founder mice, and its neuroprotective potential against ischemic brain damage was investigated. Twenty-four hours after middle cerebral artery occlusion, two DINE-Akt-transgenic mouse lines displayed reductions of the infarcted area by 35% compared to the wild-type littermate. RT-PCR assays showed a high level of transgene in response to ischemic brain damage in these lines. These results suggest that the DINE promoter is a useful promoter, which responds to neuronal insults and that the Akt-induced neuroprotective effect against ischemic damage is potent in vivo. q 2004 Elsevier Ireland Ltd. All rights reserved. Keywords: Akt; Transgenic mouse; Damage-induced neuronal endopeptidase; Endothelin Converting Enzyme Like (ECEL); Ischemia; Neuronal protection
Among the therapeutic strategies for treating cerebral ischemia, neuroprotective therapy aims to improve the viability of neurons by enhancing cell survival-promoting mechanisms. In this respect, the phosphoinositide 3-kinase (PI3-K) mediated signaling pathway, which is one of the most potent survival signaling pathways, may be a promising target of neuroprotective therapy for the treatment of ischemic neuronal damages. Along this pathway, Akt (protein kinase B; PKB), which is a serine/threonine protein kinase and activated by PI3-K, could be a pivotal molecule in mediating the cell survival signal. Akt suppresses functions of several pro-apoptotic molecules such as Bad, p53 and the forkhead transcription factors [2,3, 11], and thereby promotes the neuroprotective mechanism [7,8,12]. To evaluate the consequence of Akt activation in rescuing ischemia-induced neuronal death, we generated transgenic mice expressing Akt in neuronal cells. In this mice Akt expression is controlled by the promoter of the damage-induced neuronal endopeptidase (DINE), which is a * Corresponding author. Tel.: þ81-6-66-45-3701; fax: þ 81-6-66-453702. E-mail address: [email protected] (H. Kiyama).
unique membrane-bound metallopeptidase. DINE was initially isolated by differential display polymerase chain reaction using rat normal and axotomized hypoglossal motoneurons [6]. The most characteristic properties of DINE are neuron-specific expression and a striking expression response to a wide range of neuronal insults including brain ischemia [5]. DINE expression after middle cerebral artery (MCA) occlusion is observed in neuronal cells located near the infarction core site and most of the expressing cells are assumed to project their axons into the damaged core region. Considering this characteristic expression pattern of DINE, we speculated that the synthesis of constitutively active Akt could be locally induced in those damaged neurons after MCA occlusion, and thus this transgenic mouse would provide a useful tool to investigate the role of Akt in ischemia-related brain damage in vivo. All experimental procedures were conducted in accordance with the standard guidelines for animal experiments of the Graduate School of Medicine, Osaka City University. Transgenic mice carrying the human active form Akt gene under the control of the DINE promoter were generated. Fig. 1 shows the inserted construct of genes. In order to examine the amount of expression of the transgene, we
0304-3940/03/$ - see front matter q 2004 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2004.02.029
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N. Ohba et al. / Neuroscience Letters 359 (2004) 159–162
Fig. 1. Schematic representation of the transgene construct.
inserted the green fluorescent protein (GFP) reporter gene driven by an internal ribosomal entry site (IRES) in the downstream part of the Akt gene. GFP expression from transgenic mice was intended to monitor the synthesis of extrinsic Akt. The transgenic and wild-type (WT) littermates were generated by crossing onto a C57BL/6 background. Three lines of transgenic mice from the heterozygotes of transgenic mice originating from three different founder mice were established, maintained and used for all experiments. These three lines of transgenic mice were designated as line A – C. Unilateral permanent MCA occlusion was performed in mice (postnatal days 60 –90) as described previously [10]. Briefly, animals were anesthetized with halothane in a mixture of 70% nitrous oxide and 30% oxygen. A vertical incision was made between the orbit and external auditory canal, and subtemporal craniotomy was performed. MCA from distal to the olfactory tract was permanently occluded by bipolar electrocoagulation and transected to avoid recanalization. Then animals were allowed to recover in a cage at 37 8C for 1 h. The body temperature was maintained at 37 ^ 0.5 8C using a heating lamp and blanket. As repeated blood withdrawal is incompatible with survival, a separate group of mice (n ¼ 6 each) was used for evaluation of physiological parameters. A PE-10 femoral arterial catheter was used to sample arterial blood gases before and after MCA occlusion. Blood glucose and hematocrit were also examined. None of the physiological parameters differed significantly between transgenic and WT mice either before or after MCA occlusion (Table 1). Mice were sacrificed by decapitation at 24 h following MCA occlusion, and the brain was rapidly removed and sliced into 1 mm thick coronal sections using a brain slicer (RBS-01, Aster industries, USA). These sections were immersed in 2% 2,3,5-triphenyltetrazolium chloride (TTC) dissolved in 0.1 M phosphate buffered saline (PBS) in the dark for 30 min at 37 8C. The sections were then transferred to buffered 10% formalin for fixation overnight. The fixed sections were scanned into an image analysis system (Mac
SCOPE, Mitani corporation, Japan), and infarcts were traced at each level. The contribution of the volume increase due to the ischemic edema was corrected according to Swanson et al. [9]. All values are expressed as means ^ SD. All of the volume comparisons between transgenic and WT groups were analyzed by analysis of variance and multiple comparison test. P values of , 0.05 were considered statistically significant. RT-PCR assays were used to quantify the transgene levels in transgenic mouse lines. Total RNA was obtained from either the operated or the normal upper half of cortex at 24 h following MCA occlusion (n ¼ 3 each). Total RNA (each 0.2 mg) was converted to cDNA with superscript reverse transcriptase (Gibco/BRL) and nucleotide oligo dT18. Aliquots from the reverse transcription reaction were used for PCR amplification using primer pairs ubiquitously expressing glyceraldehyde 3-phosphatase dehydrogenase (GAPDH) as a control. The specific primers for the mRNA of Akt and GFP were used and amplified by PCR. The reaction products were separated electrophoretically on 1% agarose gel and visualized by UV photography with added ethidium bromide stain. Quantification was performed using an image scanner (GT-8200UF, EPSON, Japan) and image analysis software (Photoshop 6.0, Adobe Systems Incorporated, USA). The density of each GFP band was normalized against that of GAPDH (GFP/GAPDH). For immunohistochemistry, mice were anesthetized with diethyl ether and rapidly perfused transcardially with 0.9% sodium chloride, followed by perfusion with 4% paraformaldehyde in 0.1 M phosphate buffer (PB) at 1 day. The brain was immediately removed, and postfixed with the same solution overnight at 4 8C. The brain was then cryoprotected by storing the tissue in 30% sucrose in PB routinely, frozen in powdered dry ice and stored at 2 80 8C. Frontal sections including the ischemic region were cut at a thickness of 20 mm with a cryostat. Floating sections were pretreated with 0.3% H2O2 in Tris-buffered saline (TBS) for 30 min, rinsed three times in TBS, and incubated with TBS including 0.1% Triton X-100 containing 3% bovine serum albumin (BSA) for 1 h at room temperature (RT) to block nonspecific binding. The sections were incubated with mouse monoclonal antibody against phosphorylated Akt for 24 h at 4 8C (1:500, Cell Signaling Technology, Beverly, MA, USA) in TBS including 0.1% Triton X-100 containing
Table 1 Physiological variables Transgenic mouse
Arterial pH PaO2 (mmHg) PaCO2 (mmHg) Blood glucose (mg/dl) Hematocrit (%)
Wild-type
Pre-operation
Post-operation
Pre-operation
Post-operation
7.35 ^ 0.03 162.0 ^ 20.0 44.8 ^ 2.8 134.7 ^ 22.2 48.8 ^ 2.5
7.36 ^ 0.03 155.2 ^ 22.1 40.6 ^ 2.8 148.0 ^ 21.2 46.1 ^ 2.0
7.32 ^ 0.03 166.5 ^ 9.7 39.7 ^ 7.4 161.0 ^ 14.0 46.5 ^ 6.2
7.34 ^ 0.02 163.8 ^ 7.0 35.1 ^ 7.0 157.0 ^ 11.4 43.6 ^ 5.0
Values are means ^ SD from six animals in each group.
N. Ohba et al. / Neuroscience Letters 359 (2004) 159–162
1% BSA. They were then incubated with biotinylated antigoat IgG (1:200) for 1 h at RT, followed by incubation in ABC complex (Vector Laboratories, Inc., Burlingame, CA) for 1 h at RT. Sections were visualized by 3,30 -diaminobenzidine tetrahydrochloride –H2O2 solution and mounted onto 3-aminopropyltryethoxysilane-coated slides. All the transgenic mice belonging to the three lines appeared behaviorally normal, with no abnormalities in eating or sleeping activity. There was one apparent phenotypic difference (curly hair) in one of the pedigrees of the transgenic mouse (line B). Gross observation of transgenic mice brains did not reveal any dramatic differences in size or structure compared with the brains of their WT littermates. At 24 h after MCA occlusion, infarction size was quantified from a series of TTC stained sections (Fig. 2a). The infarct volumes of the three experimental lines were measured as mentioned before (Fig. 2b). The WT mice had a large consistent infarction of the left neocortex (15.172 ^ 5.076 mm3, n ¼ 18). The infarct sizes of line B (9.461 ^ 6.491 mm3, 37.6% reduction, n ¼ 12) and line C (9.838 ^ 6.485 mm 3, 35.2%
Fig. 2. (a) Panel of coronal slices reconstructing the forebrain 24 h after MCA occlusion representative of transgenic mouse line B (upper panel) and a WT animal (lower panel). The slices have been stained with TTC to show the area of infarction (white). (b) Determination of ischemic brain damage on WT and transgenic mice. The bar graph shows the volume of infarction in each line subjected to permanent focal cerebral ischemia. The mean infarct volumes were as follows: WT mice, 15.172 ^ 5.076 mm3, n ¼ 18; line A, 15.060 ^ 4.902 mm3, n ¼ 5; line B, 9.461 ^ 6.491 mm3, n ¼ 12; line C, 9.838 ^ 6.485 mm3, n ¼ 7. Mean infarct volumes were obtained by the summation of lesion areas in TTC staining and multiplied by slice-toslice distance. These volumes were corrected for brain swelling as described by Swanson et al. [9]. All values are expressed as means ^ SD. All of the volume comparisons between transgenic and WT lines were analyzed by analysis of variance and multiple comparison test. P , 0:05 was considered to indicate statistical significance. *P , 0:02, **P , 0:05.
161
reduction, n ¼ 7) were significantly smaller than that of the WT littermates or line A (15.060 ^ 4.902 mm3, 0.7% reduction, n ¼ 5). Several mice in line A repeatedly demonstrated relatively similar infarction size to that of WT littermates. The difference in infarct volume between WT and the transgenic mice (lines B and C) was statistically significant, as determined by analysis of variance and multiple comparison test. We examined the changes in Akt mRNAs by RT-PCR. The conditions of RT-PCR were carefully selected to prevent signal saturation. Permanent MCA occlusion resulted in up-regulation of expression of Akt mRNAs in the operated cortex (Fig. 3a). The expression level of GFP mRNA, which is a marker of exogenous expression of Akt mRNA, in the cerebral cortex was also evaluated by RTPCR analysis (Fig. 3a). We have attempted to detect the direct fluorescence of GFP on the tissue sections, however the fluorescence was not strong enough to observe with a fluorescent microscope. The expression levels of GFP mRNA in the normal cortices of line C were very low or below the level of detection, although the expression level in line B was significant even in the normal condition. At 24 h after MCA occlusion, a substantial increase of GFP mRNA in the operated side of cortices was observed in lines B and C (Fig. 3a). Fig. 3b demonstrates the magnitude of change in the relative transcript level of the GFP. Quantification of the bands by densitometry revealed that the GFP transcript level in the operated cortex of line B was 1.7 fold and of line C was 11.2 fold higher than that of nonoperated cortex. We carried out immunohistochemistry with anti-phospho-Akt antibody to investigate the expression of
Fig. 3. (a) Expression levels of Akt and GFP mRNA in transgenic mouse lines and WT detected by RT-PCR in control and operated cortex at 24 h following MCA occlusion. C, control side; O, operated side. Equivalent amounts of GAPDH were amplified from all samples, verifying standardization of conditions. (b) Quantification of results from the RTPCR shown in (a). The density of each GFP band was normalized against that of GAPDH (GFP/GAPDH). (c) Photomicrographs showing immunohistochemical localization of phospho-Akt-positive cells in the peri-infarct cortex of line B (left) and WT (right) at 1 day. Bar, 50 mm.
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active Akt protein in the brain following MCA occlusion. Phospho-Akt immunoreactive cells were seen in the periinfarct cortex of transgenic mice at 1 day after MCA occlusion, however such immunoreactivity was not seen in the WT mice (Fig. 3c). The present study demonstrates that damage-induced expression of the active Akt in mouse brain provides marked protection against ischemic damage in a model of permanent focal cerebral ischemia. Our observation adds further in vivo evidence on the functional consequences of Akt to the recent studies demonstrating the neuroprotective effects of Akt [7,8,12]. In this study, two Akt transgenic mouse lines (B and C) showed a marked increase of GFP mRNA levels in the damaged brain. One of the striking observations in this study is that the reduction of infarct volume seems to correlate with the expression level of trans-genes (AktGFP). The level of Akt-GFP mRNAs in line A after MCA occlusion might not be sufficient to provide significant tissue protection. The reason why line A failed to respond to neuronal injury is obscure, but the expression levels of the transgene could be modulated by the chromosomal position effect in a particular transgenic mouse line as the integration occurs at random chromosomal sites [1,4]. In the present study we firstly used the DINE promoter for the expression of transgene. DINE is normally expressed in a group of neurons mainly in the hypothalamus, but not in the cerebral cortex. In the transgenic mice, the expression level of transgenic Akt and GFP in normal cortex was low, but markedly induced in response to ischemic damage, which is very similar to the expression pattern of endogenous DINE. This suggests that the DINE promoter is a useful promoter for expression of molecules, which are crucial to promote rescue or neurite elongation activities in vivo. In conclusion, the protective effects of Akt under the control of the damage-specific promoter DINE after ischemic injury would provide a novel and potent therapeutic intervention in brain injuries.
Acknowledgements This study was supported in part by grants from MHLW and MEXT of Japan, Japan Brain Foundation, Novartis Foundation, and Osaka Gas Group Welfare Foundation.
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