Transgenic Mice Overexpressing Truncated trkB Neurotrophin Receptors in Neurons Show Increased Susceptibility to Cortical Injury after Focal Cerebral Ischemia

Molecular and Cellular Neuroscience 16, 87–96 (2000) doi:10.1006/mcne.2000.0863, available online at http://www.idealibrary.com on

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Transgenic Mice Overexpressing Truncated trkB Neurotrophin Receptors in Neurons Show Increased Susceptibility to Cortical Injury after Focal Cerebral Ischemia Tommi Saarelainen,* Jouko A. Lukkarinen,* ,† Susanna Koponen,* Olli H. J. Gro¨hn,* ,† Jukka Jolkkonen,* ,‡ Eija Koponen,* Annakaisa Haapasalo,* Leena Alhonen,* Garry Wong,* Jari Koistinaho,* ,‡ Risto A. Kauppinen,* ,† and Eero Castre´n* ,§,1 *A. I. Virtanen Institute, †NMR Research Group, ‡Department of Neurology and Neuroscience, and §Department of Psychiatry, University of Kuopio, Box 1627, 70211 Kuopio, Finland

It has been suggested that the increased production of endogenous BDNF after brain insults supports the survival of injured neurons and limits the spread of the damage. In order to test this hypothesis experimentally, we have produced transgenic mouse lines that overexpress the dominant-negative truncated splice variant of BDNF receptor trkB (trkB.T1) in postnatal cortical and hippocampal neurons. When these mice were exposed to transient focal cerebral ischemia by occluding the middle cerebral artery for 45 min and the damage was assessed 24 h later, transgenic mice had a significantly larger damage than wild-type littermates in the cerebral cortex (204 ⴞ 32% of wild-type, P ⴝ 0.02), but not in striatum, where the transgene is not expressed. Our results support the notion that endogenously expressed BDNF is neuroprotective and that BDNF signaling may have an important role in preventing brain damage after transient ischemia.

Brain-derived neurotrophic factor (BDNF) is abundantly expressed in several brain areas, including hippocampus and cerebral cortex, but the physiological function of BDNF in adult brain is not clear. Expression of BDNF is dynamically regulated in response to neu1 To whom correspondence and reprint requests should be addressed at A.I. Virtanen Institute, University of Kuopio, P.O. Box 1627, 70211 Kuopio, Finland. Fax: ⫹358-17-163 030. E-mail: [email protected].

1044-7431/00 $35.00 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved.

ronal activity (Gall, 1992; Lindholm et al., 1994; Thoenen, 1995) and increasing evidence is connecting activation of trkB receptors by BDNF to neuronal plasticity and synapse stabilization (Thoenen, 1995; McAllister et al., 1999). In addition, BDNF expression is also stimulated by brain insults, such as ischemia and epileptic seizures (Lindvall et al., 1994; Gall et al., 1997). It has been speculated that increased expression of BDNF in these pathological conditions would protect vulnerable neurons (Lindvall et al., 1994), but there is little experimental evidence to support this hypothesis, in part because knock-out mice for BDNF and trkB die before adulthood (Klein et al., 1993; Ernfors et al., 1994; Jones et al., 1994). Expression of endogenous BDNF mRNA and protein are increased by global (Lindvall et al., 1992, 1996; Takeda et al., 1993; Kokaia et al., 1994, 1996; 1998; Matsushima et al., 1998; Tsukahara et al., 1998) and focal ischemia (Kokaia et al., 1995; Arai et al., 1996). After a transient occlusion of the middle cerebral artery (MCA), BDNF mRNA is increased mainly in neurons in the areas adjacent to the necrotic core (Kokaia et al., 1995; Arai et al., 1996). Increased BDNF expression is correlated with reduced vulnerability after ischemia (BorisMoller et al., 1998; Matsushima et al., 1998), whereas BDNF reduction correlates with increased damage (Uchino et al., 1997; Yamasaki et al., 1998). Moreover, infusion of BDNF into brain or even systemically dur-

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88 ing and after the induction of global or focal ischemia reduces ischemia-induced neuron loss (Beck et al., 1994; Cheng et al., 1997; Schabitz et al., 1997; Ferrer et al., 1998; Kiprianova et al., 1999; Mitchell et al., 1999; Wu and Pardridge, 1999). These data suggest that the increase in endogenous BDNF in response to ischemia would protect vulnerable neurons. TrkB receptor tyrosine kinase, the receptor for BDNF and neurotrophin-4, is alternatively spliced into a fulllength tyrosine kinase containing isoform (trkB.TK) and two truncated forms which lack the tyrosine kinase domain and contain short unique intracellular tails (trkB.T1 and T2) (Klein et al., 1990; Middlemas et al., 1991). As BDNF signaling is initiated by a trkB dimerization and transphosphorylation, truncated receptors are expected to act as dominant negative receptors and inhibit the signal transduction of the full-length receptor when expressed in the same cell. Indeed, introduction of trkB.T1 to cells which express trkB.TK has been shown to inhibit BDNF signaling (Eide et al., 1996; Ninkina et al., 1996; Li et al., 1998). Furthermore, trkB receptor bodies, soluble fusion molecules with trkB extracellular domain fused to the IgG-Fc region, are routinely used to inhibit trkB signaling (Shelton et al., 1995). In an attempt to investigate the physiological roles of endogenous neurotrophins in adult brain we have produced a mouse that overexpresses dominant negative trkB.T1 receptors in postnatal neurons. We have here exposed these mice to transient focal cerebral ischemia using the MCA occlusion model and observed that transient ischemia produces a significantly larger lesion in the cerebral cortex of transgenic mice as compared to wild-type littermates. These results support the hypothesis that endogenous BDNF protects CNS neurons from ischemic injury.

RESULTS We have produced a mouse with reduced trkB signaling in adult brain by overexpressing truncated trkB receptors in postnatal neurons. We chose Thy1 minigene to drive the expression of trkB.T1 (Fig. 1a), because previous reports have shown that this promoter is active in postnatal neurons (Ingraham and Evans, 1986; Aigner et al., 1995). Transgene expression remained at the level of about 4-fold to the endogenous trkB.T1 expression until about postnatal day 16, after which it was strongly increased by postnatal day 18 to a level that represents about 20-fold overexpression compared to the endogenous truncated trkB mRNA and remained stable thereafter (Fig. 1b). In situ hybridization revealed

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FIG. 1. Transgenic mice overexpressing trkB.T1 in neurons. (a) Construct used for pronucleus injections. cDNA coding for Flag-tagged trkB.T1 was inserted into the Thy1 minigene. (b) Northern blot from brains of wild-type (Wt) and transgenic (Tg) mice of different ages (between P7 and P28) hybridized with a probe against the extracellular portion of the trkB mRNA. Bands corresponding to the endogenous trkB.T1 mRNA (T1) and the trkB.T1 mRNA produced by the transgene (tg) are indicated. (c) In situ hybridization of a wild-type (left) and transgenic (right) mouse brain at two different rostrocaudal levels hybridized with a probe recognizing trkB extracellular regions. cx, cortex; hi, hippocampus; st, striatum; th, thalamus. Magnification, 8⫻.

an uneven distribution of the transgene mRNA in brain (Fig. 1c). In all brain areas, however, transgene expression appeared to be confined to neurons. High levels of transgene mRNA were found in layers II, III, and V of the cerebral cortex, in the pyramidal cell layer of all hippocampal subfields, and in thalamus. In contrast, no transgene expression was detected in striatum (Fig. 1c). Immunostaining with anti-Flag antibody confirmed neuronal localization and that the transgene mRNA was translated into protein (Fig. 2A). Particularly strong expression of the trkB.T1–Flag fusion protein was seen in the pyramidal neurons in the cortical layer V in

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FIG. 2. Expression of the transgene and inhibition of trkB signaling in cortical neurons. Upper panel: Immunohistochemical staining with anti-Flag antibody showing the expression of trkB.T1-Flag fusion protein in pyramidal neurons in cortical layer V in transgenic (A) and wild-type mouse (B). Magnification 40⫻. Lower panel: Phospho-trk immunohistochemistry in layer V cortical neurons in wild-type (C) and transgenic mouse (D). Magnification, 100⫻.

transgenic mice (Fig. 2A), whereas only background staining was seen in wild-type brain, as expected (Fig. 2B). It was important to demonstrate that overexpression of trkB.T1 inhibits the signaling of trkB ligands. The fact that the transgene was not expressed early enough in cultured hippocampal or cortical neurons derived from transgenic mice (T.S. and E.C., unpublished results) prevented us from testing the effects of trkB.T1 in these culture systems. We therefore incubated acute cortical slices from brains of transgenic and wild-type mice in the presence of BDNF (100 ng/ml) for 30 min and immunostained fixed sections with an antibody that recognizes the activated, phosphorylated form of trkB.TK isoform (Segal et al., 1996). In wild-type BDNF-

treated cortical slices, antibody clearly stained numerous cell bodies in the layer V of the cortex (Fig 2C). In contrast, very few clearly stained cell bodies could be seen in BDNF treated slices derived from trkB.T1 transgenic mice (Fig. 2D). Counting of antibody-stained cell bodies confirmed that significantly larger number of cell bodies were stained with phospho-trk antibody in slices derived from wild-type than transgenic mice (40 ⫾ 2.5 and 7.6 ⫾ 1.9 cell bodies in wild type and transgenic slices, respectively, P ⬍ 0.001). These data demonstrate that overexpression of trkB.T1 in transgenic mice significantly inhibited the ability of BDNF to activate trkB receptors in cortical neurons. Focal cerebral ischemia has been shown to induce the expression of BDNF mRNA in rat cortex and hippocam-

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FIG. 3. Expression of BDNF mRNA after transient focal ischemia in a wild-type mouse. Upper panel: sham-operated mouse (CON) and a mouse brain 3 h after a 45-min MCA occlusion (ISCH). Lower panel: Basal BDNF mRNA expression in wild-type (WT) and transgenic mouse brain (TG).

pus (Kokaia et al., 1995). As shown in Fig. 3b, BDNF mRNA was increased in the dentate gyrus, medial CA1 region, as well as the parietal and piriform cortex adjacent to the ischemic core region 3 h after 45 min of MCA occlusion. This pattern is similar to that described in rats (Kokaia et al., 1995). The basal expression of BDNF mRNA was not significantly different between wildtype and transgenic mice (Figs. 3c and 3d). In order to test the hypothesis that increased BDNF expression following ischemia might be neuroprotective, we investigated the susceptibility of transgenic and wild-type mice to a short ischemia with reperfusion by exposing them to transient MCA occlusion. A relatively short occlusion time of 45 min was used to detect exacerbation of ischemic damage by the transgene. Initial experiments with mice from two lines 186 and 188 (n ⫽ 3 ⫹ 3 and n ⫽ 6 ⫹ 7 for wild-type littermate ⫹ heterozygous transgenic mice, respectively) showed

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similar results (data not shown). Line 188 was used in further experiments. Physiological variables in separate groups of mice exposed to MCA are shown in Table 1. A mild acidosis and hypercarbia was evident during ischemia in both groups of mice, but there were no significant differences between the groups, a finding also reported in other mouse models (Hata et al., 1998). Diffusion magnetic resonance imaging (MRI) showed that a similar degree of D av (trace of the diffusion tensor, see Experimental Methods) reduction occurred in both transgenic and wild-type mice during MCA occlusion (Table 1; Fig. 4a). The damage produced by a 45-min MCA occlusion and 24-h reperfusion was significantly larger in the cerebral cortex of transgenic mice when compared to their wild-type littermates (18.1 ⫾ 2.8 mm 3 vs 8.9 ⫾ 0.9 mm 3, P ⫽ 0.02) (Figs. 4b and 5). In contrast, striatal lesion size did not show significant differences between transgenic and wild-type mice (9.5 ⫾ 0.5 mm 3 and 9.0 ⫾ 0.9 mm 3, respectively, Figs. 4b and 5), which is to be expected, since the transgene is not expressed in striatum (Fig. 1c). Although hippocampus is not directly vascularized by MCA, we observed a variable degree of hippocampal damage in most mice in both experimental groups. There was a trend towards a larger lesion size in the hippocampus in transgenic mice than in controls (3.0 ⫾ 1.0 mm 3 vs 1.9 ⫾ 0.9 mm 3; Fig. 5), but the difference was not statistically significant, mainly due to a large variation in the hippocampal lesion sizes in both groups. No differences in the vascular patterns were seen between the two genotypes by using an ink perfusion to visualize the vasculature (not shown) and diffusion imaging did not show any significant differences between groups (similar D av values, Table 1), which strongly suggests that blood vessel anomalies in transgenic mice do not explain the result. Taken together, these findings strongly argue that overexpression of the dominant negative trkB receptor exacerbates the damage produced by ischemia and reperfusion.

DISCUSSION BDNF is dynamically expressed after brain lesions and it has been suggested that this increased expression and release would protect damaged neurons from death (Lindvall et al., 1994; Gall et al., 1997). It has been difficult to test this hypothesis in vivo, however, partially because knock-out mice for BDNF (Ernfors et al., 1994; Jones et al., 1994) and trkB (Klein et al., 1993) die before adulthood. TrkB.T1 isoform has been shown to inhibit BDNF signaling when it is expressed in the same

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Cerebral Ischemia in trkB.T1 Overexpressing Mice

TABLE 1 Physiological Parameters during Ischemia in Wild-type and Transgenic Mice Wild-type (n ⫽ 7)

Transgenic (n ⫽ 10)

Variables

Before

During MCA occlusion

Before

During MCA occlusion

MABP (mm Hg) pH PaCO 2 (mm Hg) PaO 2 (mm Hg) Temperature (°C)

93 ⫾ 2 7.21 ⫾ 0.05 59 ⫾ 4 105 ⫾ 9 37.0 ⫾ 0.19

80 ⫾ 6 7.19 ⫾ 0.09 55 ⫾ 5 101 ⫾ 5 37.0 ⫾ 0.06

103 ⫾ 5 7.20 ⫾ 0.07 54 ⫾ 6 108 ⫾ 8 36.7 ⫾ 0.19

86 ⫾ 6 7.22 ⫾ 0.09 49 ⫾ 4 115 ⫾ 7 36.9 ⫾ 0.08

D av (10 ⫺3 mm 2/s)

Contralateral

Ipsilateral

Contralateral

Ipsilateral

0.80 ⫾ 0.02

0.54 ⫾ 0.03

0.79 ⫾ 0.09

0.53 ⫾ 0.01

Note. Mean arterial blood pressure (MABP) and blood samples were obtained before and 15 min from the onset of MCA occlusion. D av data are from contralateral and ipsilateral hemispheres. Data shown as means ⫾ SEM. There were no statistically significant differences between groups (Student’s t test).

cell together with trkB.TK receptor either by acting as a dominant negative receptor and preventing the activation of full-length trkB isoform or by trapping the ligand and preventing it from interacting with the fulllength receptor (Biffo et al., 1995; Eide et al., 1996; Ninkina et al., 1996; Li et al., 1998). We have produced a mouse which expresses high levels of trkB.T1 in neurons only postnatally in order to avoid the deleterious effects in the peripheral nervous system that trkB knock-out mice succumb to (Klein et al., 1993). The lines that we have produced fulfill this criteria: transgene expression is strongly induced only during the third postnatal week and inhibits the ability of BDNF to activate trkB receptors in cortex of adult mice, but the mice survive and are fertile and can therefore be used to investigate the role of endogenous trkB ligands in brain lesions. Minichiello et al. (1999) have recently published a forebrain-specific conditional trkB knock-out mouse, which survives to adulthood and shows reduced spatial learning capacity. We have also observed that the longterm memory of trkB.T1 overexpressing mice is impaired, although to a lesser extent than that of the forebrain-specific trkB mutant mice (Saarelainen et al., 2000). It would be of interest to study the response of the conditional trkB mutant mice to brain lesions such as ischemia. Focal cerebral ischemia increases BDNF mRNA expression in rat hippocampus as well as in the cortical areas surrounding the ischemic core, but not within the core itself (Kokaia et al., 1995; Arai et al., 1996), and we have confirmed that similar distribution of BDNF mRNA increase also takes place in mice 3 h after transient MCA occlusion. This pattern suggests that in-

creased expression of endogenous BDNF may support the survival of less severely damaged neurons in the regions bordering the necrotic core. Correlative evidence suggests that in situations where the normal increase in cortical BDNF is not taking place, neurons are more susceptible to ischemia (Uchino et al., 1997; Yamasaki et al., 1998). Furthermore, mutations in the trkB gene have been found in a stroke-prone rat strain (Kageyama et al., 1996). We show here that cortical ischemic lesion is significantly larger in transgenic mice overexpressing trkB.T1 in neurons. Together these observations strongly support the hypothesis of the neuroprotective role of endogenous BDNF in cerebral ischemia. The degree of ischemic lesion appeared to correlate with the expression of the transgene in different brain areas. In the cerebral cortex, where high levels of transgene are expressed, trkB.T1 overexpressing mice suffer a significantly larger damage after the exposure to a transient 45-min occlusion of MCA when compared to wild-type littermates. In contrast, lesion volumes were similar in both transgenic and wild-type mice in striatum, where the transgene is not expressed. In hippocampus, increased damage was observed in the majority of transgenic mice, but some mice in both groups had only a small hippocampal lesion or no lesion at all, which is most probably due to the different degree of dependence of hippocampus on the blood supply through MCA in individual mice. Although the mean lesion size was larger in transgenic mice than in controls, the difference was not statistically significant due to high variation. In our preliminary experiments, however, we observed a significantly increased damage in

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FIG. 4. Effects of MCA occlusion in trkB.T1 transgenic mice (TG) and wild-type littermates (WT). (a) Representative trace of diffusion tensor images showing reduced diffusion (in blue) in the MCA territory at two different rostrocaudal mouse brain slices (approximate section levels: upper panel, bregma ⫹0.15 mm; lower panel, bregma ⫺2.7 mm). (b) The damaged tissue at 24 hours after MCA occlusion displayed as hyperintensity in T 2-weighted MR-images at two representative imaging slices (approximate section levels as in a). Neuronal damage appears as bright areas. cx, cortex; hi, hippocampus; st, striatum; th, thalamus.

hippocampus of transgenic compared to wild-type mice (data not shown), but because a different method was used for damage assessment in the preliminary experiments and experiments shown here, the data from both groups could not be combined. Nevertheless, the apparent correlation between the level of transgene expression and the size of ischemic lesion further supports the direct role of trkB signaling in the protection against ischemic damage. It has been suggested that trkB.T1 may mediate sig-

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naling on its own. BDNF treatment of cell lines expressing truncated trkB isoforms has been reported to produce an acidification response indicative of signal transduction (Baxter et al., 1997), but the nature of this signal transduction and whether it takes place in neurons is unknown. We have recently shown that transfection of trkB.T1 into cell lines induces a morphological response (Haapasalo et al., 1999), but we have not detected such morphological changes in cultured neurons after transfection with trkB.T1 (A.H. and E.C., unpublished observation). Nevertheless, it is possible that the mice which overexpress trkB.T1 receptors may, in addition to the inhibition of the trkB.TK receptor mediated responses, also display some effects mediated directly by the trkB.T1 isoform itself and these effects might contribute to the exacerbation of the ischemic lesion observed in transgenic mice. Use of mice in focal cerebral ischemia induced by MCA occlusion is not as common as that of rats. From the point of view of damage interpretation, it is of central importance to verify the presence of ischemia. We used MRI to identify the animals with similar degrees of water diffusion reduction (⬎0.20 ⫻ 10 ⫺3 mm 2/s) at a given time point of MCA occlusion. A decrease in absolute diffusion coefficient of water is an inevitable consequence of a reduction of blood flow to ⬍20 ml/100 g/min (Moseley et al., 1990; Kohno et al., 1995; Miyabe et al., 1996). The threshold of diffusion drop used here has been shown to indicate cerebral blood flow below ischemic energy failure level in a cat model of MCA occlusion (Miyabe et al., 1996). Thus, we are confident that the mice used in lesion size analysis 24-h after MCA occlusion suffered genuine ischemia and that the duration of ischemia is comparable between the two groups. Another important aspect of MRI is that bias caused

FIG. 5. Relative lesion volumes in brains of transgenic mice (TG, n ⫽ 6) and wild-type littermates (WT, n ⫽ 5) shown as percentage ⫾ SEM of wild-type values. Lesions in cortex, striatum, and hippocampus were quantitated from T 2-weighted MR-images (*P ⫽ 0.018, t test).

Cerebral Ischemia in trkB.T1 Overexpressing Mice

by complications, such as bleeding due to the thread introduction during MCA occlusion, could be excluded from the analysis. Lesion volumes are analyzed a day after MCA occlusion due to the fact that a sufficient number of survivors is obtained, and this time point is commonly used in mouse models of focal cerebral ischemia. Lesion volumes tend to grow during the first three days after MCA occlusion in mice (Iadecola et al., 1997). The present experimental design cannot exclude the possibility that the difference between animal groups in cortical lesion volumes would have been even larger at later time points than that determined at 24 h (Eliasson et al., 1997). Significant factors determining damage development include the physiological parameters quantified from a separate group of mice (Table 1). A moderate respiratory acidosis developed in both animal groups, which appears to be a common consequence of MCA occlusion in spontaneously breathing mice (Hata et al., 1998). However, since the degree of acidosis was similar in both animal groups it is unlikely that it would influence the difference in the cortical lesion size between wild-type and transgenic mice. In conclusion, we have shown that mice that overexpress truncated trkB receptors in neurons suffer from a much greater cortical damage after a transient cerebral ischemia than their wild-type littermates. This indicates a significant role for the endogenous BDNF in neuronal survival after transient focal ischemia. Finally, trkB.T1 transgenic mice may also be useful to elucidate the role of endogenous neurotrophins in other pathological as well as physiological conditions.

EXPERIMENTAL METHODS The expression construct used in the production of the transgenic mice is shown in Fig. 1a. The full-length trkB.T1 cDNA was tagged N-terminally with an eight amino acid FLAG peptide as previously described (Haapasalo et al., 1999) and inserted into murine Thy 1.2 expression cassette, which directs transgene expression to postnatal neurons (Ingraham and Evans, 1986; Aigner et al., 1995). Transgenic mice were generated by pronucleus injection of this construct into embryos from CD2F1 (BALB/c ⫻ DBA/2) female mated with CD2F1 males. Transgenic founder mice were identified using genomic DNA extracted from tail pieces by Southern blot analysis. In order to identify descendants, PCR was also used with Thy 1 and trkB-specific primers. Three independent lines were produced, of which two, with similar temporal and spatial transgene expression patterns were used in these experiments. Het-

93 erozygous mice were viable and fertile and preliminary histological analysis did not show any gross brain abnormalities (T.S. and E.C., unpublished results). Heterozygous mice and their wild-type littermates were used in all these experiments. The production of transgenic mice and all the animal experiments were done in accordance to the guidelines of the Society for Neuroscience and were accepted by the experimental animal ethics committee of the University of Kuopio. Northern blot analysis was performed with total RNA isolated from the brains of mice aged 7 to 28 days by Trizol reagent (Life Technologies), separated (10 ␮g of RNA per lane) on formaldehyde containing 1% agarose gel and transferred to a nylon membrane (Hybond, Amersham). The filters were prehybridized for 1 h at 65°C in 0.5 M NaH 2PO 4 (pH 7.0), 1 mM Na 2EDTA, 7% SDS, 0.5% BSA, and hybridized overnight at 65°C in the same buffer together with a [␣- 32P]dCTP-labeled trkB cDNA probe, which recognizes all the isoforms of trkB. In situ hybridization was performed essentially as described before (Lindholm et al., 1993). Frozen sections (12 ␮m thick) were postfixed in 4% buffered paraformaldehyde, treated with acetic anhydride, and hybridized overnight at 58°C with [ 33P]-UTP-labeled cRNA probes recognizing mRNAs for mouse BDNF or the extracellular domains of mouse trkB. After hybridization, the sections were incubated with RNaseA (Sigma), washed under increasing stringency up to 0.1⫻ SSC at 60°C, dehydrated, and exposed to Hyperfilm ␤-Max (Amersham) for 3 days (trkB) or 2 weeks (BDNF). For immunohistochemistry, mice were anesthetized with pentobarbital, perfused with 4% buffered paraformaldehyde, and brains were postfixed for 2 h in the same fixative. Vibratome sections (50 ␮m) were first washed (PBS, pH 7.4), blocked (10% serum, 0.5% Triton X-100 in PBS), and incubated with anti-FLAG BioM2 antibody (Kodak) (1 ␮g/ml, 0.5% Triton X-100, 1% serum in PBS) overnight at room temperature (RT). After primary antibody incubation sections were washed and incubated with secondary antibody (1% serum in PBS, 1 h, RT). Bound antibody was visualized with 3,3⬘diaminobenzidine as peroxidase substrate using a Vectastain kit (Vector Laboratories). For visualization of vasculature, mice were perfused with indian ink, brains were removed and photographed from four sides and the course of the arterial circle and the middle cerebral artery was compared between transgenic and wild-type mice. In order to verify that the transgene inhibits trkB signaling, animals (adult transgenic heterozygotes and wild-type littermates, n ⫽ 3 ⫹ 3) were quickly killed with carbon dioxide and decapitated. Brains were dis-

94 sected, cooled, and sliced in ice-cold PBS to 500-␮m coronal sections with a vibrating tissue slicer (Vibroslicer, Campden Instruments, Sileby, UK). Slices were preincubated in oxygenated (95% O 2/5% CO 2) PBS for 2 h at 37°C. After preincubation, brain slices were incubated in oxygenated PBS containing 1 mM sodium orthovanadate (PBSV) with BDNF (100 ng/ml, Peprotech) at 37°C for 30 min. Slices were postfixed in 4% paraformaldehyde at 4°C in PBSV and cryoprotected in 20% sucrose in PBSV. Frozen coronal sections (30 ␮m) were cut from slices and sections were mounted in PBSV onto Superfrost (Corning) slides, dried, and processed immediately for immunohistochemistry. Immunohistochemistry was performed with an anti-phospho-Trk antibody (New England Biolabs) essentially as described by Binder et al. (1999). FITC-conjugated antirabbit IgG (1:200, Sigma, MO) was used as a secondary antibody. Quantification of phospho-trk-stained cells was performed under fluorescence microscope. Phospho-trkpositive layer V pyramidal cells with typical pyramidal cell morphology were counted at equivalent coronal levels (bregma ⫺2.70 mm) from sections of transgenic mice and wild-type littermates. Cells from 24 visual fields were counted from both groups under 20⫻ objective. Transient MCA occlusion was produced by an intraluminal thread method (Longa et al., 1989). In a set of male mice (wild-type littermates: n ⫽ 5, 26 –32 g; heterozygous transgenics: n ⫽ 6, 27–30 g, age 5–11 months) lesion size was assessed using MRI. Mice were initially anesthetized with 4% halothane and maintained on 0.8 –1% halothane (70% N 2O/30% O 2). The right common carotid artery was exposed and external carotid artery was ligated. Monofilament nylon thread (5-0) was inserted into the internal carotid artery up to the anterior cerebral artery and fixed at this position for 45 min. Body temperature was maintained by a feedbackcontrolled heating blanket at approximately 37.0°C and blood pressure and body temperature (rectal) were monitored (Table 1). Blood pH, PaCO 2, and PaO 2 were measured before and 15 min after the beginning of the occlusion in a separate group of mice (7 wild-type and 10 transgenic mice, Table 1). Hypothermic animals and mice with subarachnoidal hemorrhage were excluded from further analysis. Diffusion magnetic resonance imaging (MRI) was used to assess the presence of ischemia during MCA occlusion. Male animals were placed in a custom-built holder with ear bars and teeth fixation securing a stable positioning. Animals were taken into a horizontal 4.7T magnet (Magnex Scientific Ltd., Abdington, UK)

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equipped with actively shielded gradients (maximal amplitude 17 G/cm) interfaced to a Surrey Medical Imaging console (Guildford, UK). Surface coil (diameter 22 mm) was used for signal transmission and reception. ⫽ The trace of the diffusion tensor (D av ⫽ 31 TraceD ) was quantified using a spin echo-based method with acquisition parameters as follows: repetition time (TR) of 1500 ms, echo time (TE) of 52 ms, field of view (FOV) of 20 mm, slice thickness (ST) of 1 mm, data matrix of 128 ⫻ 64 (zero filled to 128 square prior to Fourier transformation), two dummy scans, two averages per view. Four bipolar gradients of 4 ms in each orthogonal direction were used to weight the images for D av (Mori and van Zijl, 1995) (b values of 35 and 1260 s/mm 2). Absolute D av images were computed on a pixel-by-pixel basis in Matlab routine (Mathworks Inc, Natlik, MA). Absolute D av images were acquired 15 min after introducing the occluder thread. D av decrease more than 0.20 ⫻ 10 ⫺3 mm 2/s relative to the contralateral hemisphere were used as ischemia thresholds, since it has been recently shown that D av drop by ⬎25% indicates cerebral blood flow levels below level of ischemic energy failure (Miyabe et al., 1996). Lesion volumes in the cortex, striatum and hippocampus of transgenic (n ⫽ 6) and wild-type mice (n ⫽ 5) at 24 h after reperfusion were determined from T 2-weighted images as previously described (Lukkarinen et al., 1997). MRI acquisition parameters for these images were as follows: TR of 2000 ms, TE of 55 ms, data matrix of 256 ⫻ 128 (zerofilled prior to Fourier transformation), FOV of 20 mm, ST of 0.7 mm, 12 consecutive slices, four scans per view. Data are shown as means ⫾ SEM and Student’s t test was used for statistical analysis.

ACKNOWLEDGMENTS We are grateful to Laila Kukkonen, Tuula Reponen, Jukka Korhonen, Nanna Huuskonen, and Anna-Liisa Gidlund for excellent technical assistance, Dr. Mart Saarma for trkB.T1 cDNA, and Dr. Pico Caroni for Thy1.2 minigene. This work was supported by grants from European Union Biotech program (PL980259), Academy of Finland, Sigrid Juselius Foundation, and J. and R. Ahokas Foundation.

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Cerebral Ischemia in trkB.T1 Overexpressing Mice

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