Journal of Cerebral Blood Flow and Metabolism 20: 139-144 © 2000 The International Society for Cerebral Blood Flow and Metabolism Published by Lippincott Williams & Wilkins, Inc., Philadelphia

Ischemic Brain Damage in Mice After Selectively Modifying BDNF or NT4 Gene Expression

Matthias Endres, *Guoping Fan, Lorenz Hirt, Masazumi Fujii, Kohji Matsushita, *Xin Liu,

*Rudolf Jaenisch, and Michael A. Moskowitz

Stroke and Neurovascular Regulation Laboratory, Massachusetts General Hospital, Harvard Medical School, Boston; and

*Whitehead Institute Jor Biomedical Research, Massachusetts Institute of Technology, Cambridge, Massachusetts, U.S,A.

Summary: The neurotrophins and the tyrosine kinase (Trk) B receptor may play a protective role in the pathophysiology of cerebral ischemia. In this study, the authors investigated wheth­er reducing endogenous expression of TrkB-binding neuro­trophins modifies the susceptibility to ischemic injury after I-hour middle cerebral artery occlusion followed by 23 hours of reperfusion in a filament middle cerebral artery occlusion model. Mice lacking both alleles for neurotrophin-4 (nt4-1-) or

Neurotrophins belong to a family of growth factors that are required for the survival and maintenance of neurons in the peripheral and central nervous system. Members of this family include nerve growth factor (Klein et aI. , 1991a), brain-derived neurotrophic factor (BDNF) (Leibrock et aI. , 1989), neurotrophin-3 (NT3) (Emfors et aI. , 1990), and neurotrophin-4/5 (NT4) (Ip et aI., 1992). The neurotrophins specifically bind to both a receptor tyrosine kinase (TrkA, TrkB, or TrkC) (Klein, 1991a,b; Lamballe, 1991; Soppet, 1991; Squinto et aI. , 1991) and a common low-affinity p75 receptor (Hall­book et aI. , 1991; Lee et aI. , 1994).

Neurotrophins may play a protective role in several neurodegenerative diseases, brain trauma, epilepsy, and

Received March 15, 1999; final revision received June 25, 1999; accepted June 30, 1999.

M. Endres and G. Fan contributed equally to this work. Studies were supported by grants from the National Institutes of

Health (NS10828 to M.A. Moskowitz), Amgen (R. Jaenisch), the Medical Foundation (G. Fan), the Deutsche Forschungsgemein­schaft (En 343/1-1 to M. Endres), the Schweizerische Stiftung fur Medizinisch-Biologische Stipendien (L. Hirt), and the SICPA founda­tion (L. Hirt).

Address correspondence and reprint requests to Michael A. Moskowitz, Stroke and Neurovascular Regulation Laboratory, Massa­chusetts General Hospital, Harvard Medical School, 149 13th Street, Room 6403, Charlestown, MA 02129, U.S.A.

Abbreviations: BDNF, brain-derived neurotrophic factor; MCAO, middle cerebral artery occlusion; NT, neurotrophin; Trk, receptor ty­rosine kinase.

139

deficient in a single allele for brain-derived neurotrophic factor (bdnJ+/-) exhibited larger cerebral infarcts compared to wild­type inbred 129/SVjae mice (68% and 91 %, respectively, com­pared to controls). Moreover, lesions were larger (21 %) in nt4-1- mice after permanent middle cerebral artery occlusion. Hence, expression of both NT4 and BDNF, and by inference the TrkB receptor, confers resistance to ischemic injury. Key Words: Neurotrophins-Cerebral ischemia-BDNF-NT4.

cerebral ischemia (Merlio et aI. , 1993; Lindvall et aI. , 1994; Kokaia et aI. , 1995, 1996). For example, the ex­ogenous administration of BDNF or NT4 which both bind to TrkB reduced brain damage in several animal models of cerebral ischemia (Beck et aI. , 1994; Tsuka­hara et aI. , 1994; Chan et aI., 1996; Alexi et aI., 1997; Cheng et aI. , 1997; Schiibitz et aI. , 1997). The precise role of endogenous TrkB receptor agonists in the devel­opment of ischemic brain injury, however, remains poorly understood. In this study we detennined if a de­ficiency in endogenous BDNF or NT4 renders the brain more susceptible to ischemic injury. We compared lesion volume and neurologic outcome after middle cerebral artery occlusion (MCAO) in mice lacking either one bdnf allele or both al1eles of nt4.

MATERIALS AND METHODS

Knockout mice The generation of neurotrophin-4 knockout (ntrl-) has been

described before (Liu et aI., 1995). NT4-1- mice are viable and fertile and can be crossed to produce offspring (Liu et aI., 1995). Brain-derived neurotrophic factor knockout mice (bdnr'-) die within 2 to 3 weeks after birth, but heterozygote (bdnjl- ) mice are healthy and fertile and were generated and genotyped as described (Emfors et a!., 1994). All nt4-1-, bdnj"I-, and wild-type control mice carried a pure inbred 129/ SVjae background. NT4-1- and bdnj"l- mice have normal ap­pearing brain vasculature and morphology.

140 M. ENDRES ET AL.

Northern blot Total RNA was extracted from the cortex, subjected to gel

electrophoresis, and blotted to the membrane in a standard procedure (Ernfors et aI., 1994). Membranes were subsequently hybridized with p32-labeled radioactive nt4 and bdnf probes and exposed to x-ray film (Kodak, Rochester, NY, U.S.A.) for autoradiography.

Model of cerebral ischemia All animal experiments were performed in accordance with

the National Institutes of Health (NIH) guide for the care and use of laboratory animals (NIH publications No. 80-23) and institutional guidelines (Massachusetts General Hospital and Massachusetts Institute of Technology). Gender-matched mice (age, 1.5 to 4 months; body weight, 18 to 26 g) were first anesthetized with the use of halothane (1.5% for induction and 0.8% for maintenance) in 70% nitrous oxide (N20) and 30% oxygen (02) administered with a face mask. Focal cerebral ischemia was produced as described (Huang et a!., 1994; En­dres et a!., 1997, 1998b). Briefly, the left middle cerebral artery was occluded with an 8-0 silicone-coated monofilament. For transient ischemia, the filament was withdrawn after 1 hour of ischemia. For permanent ischemia, the filament was kept in place until the animal was killed at 24 hours. In all animals, regional cerebral blood flow was measured by means of a flex­ible probe and laser-Doppler monitoring (Perimed, Smithtown, NY, U.S.A.) as described previously (Huang et a!., 1994; En­dres et a!., 1998a,b).

Physiologic parameters Recording of physiologic parameters was essentially per­

formed as previously described (Huang et a!., 1994; Endres et a!. 1998a,b). Briefly, rectal temperature was controlled and kept constant at 37 ± 0.5°C by means of a feedback temperature control unit (FHC, Brunswick, ME, U.S.A.) and a heating lamp for the duration of ischemia until I hour after reperfusion. After 24 hours, rectal temperature was again measured, and no dif­ferences were found between groups «O.4°C, P > 0.05). In randomly assigned animals, the left femoral artery was cannu­lated using a PE-IO catheter for arterial blood pressure moni­toring and blood withdrawal. Arterial blood samples were ana­lyzed for pH and partial pressure of oxygen and carbon dioxide (Corning 178, Ciba-Corning Diagnostics, Medfield, MA, U.S.A.).

Determination of infarct size Animals were killed at 24 hours by an overdose of pento­

barbital (200 mg/kg, administered intraperitoneally). Subse­quently, brains were sectioned coronally (2 mm) using a matrix (RBM-2000C, Activational Systems, Ann Arbor, MI, U.S.A.) and stained with 2% 2,3,5-triphenyltetrazolium chloride. In­farction volume was measured quantitatively according to Swanson et al. (1990) or using an indirect method that corrects for brain swelling as described (Endres et a!., 1998b).

Histology Animals were killed at 24 hours as above, and brains were

frozen in liquid nitrogen vapor. Frozen coronal sections 1,500 fLm in distance and 14 fLm thick were cut on a cryostat (Mi­crotome cryostat, Model HM505E, Microm, Walldorf, Ger­many), thaw-mounted on precleaned glass slides, and stained with hematoxylin and eosin according to a standard protocol.

Immunohistochemistry Thawed tissue sections were dried completely and postfixed

in absolute ethanol at -20°C for 10 minutes. After several washes in 0.1 mollL phosphate-buffered saline, the sections were incubated with 10% normal goat serum containing 0.3%

J Cereb Blood Flow Metab, Vol. 20, No.1, 2000

H202 for I hour at room temperature. For immunohistochem­ical staining, sections were incubated with a monoclonal anti­body against neuronal nuclear protein (NeuN, Chemicon Inter­national Inc., CA, U.S.A., 1 :300) at 4°C overnight. After wash­ing in phosphate-buffered saline, the sections were incubated with bodipy fluorescein (BODIPY-FL) conjugated goat anti­mouse IgG secondary antibody (Molecular Probes, Eugene, OR, U.S.A.) for I hour at room temperature. After washing in phosphate-buffered saline, sections were dehydrated in ascend­ing ethanol series, immersed in xylene, and coverslipped with Permount (Fisher Scientific, Pittsburgh, PA, U.S.A.)

Neurologic deficits Scoring of sensorimotor deficits was performed by an ob­

server nai've to the genotype using a scale from 0 (no deficit) to 3 (severe) as described (Huang et a!., 1994; Endres et a!., 1998a,b).

Statistical analysis All experiments were performed in a blinded fashion. Values

are presented as mean ± standard deviation (SD). Statistical comparisons were performed by unpaired Student's t-test (in­farcts), ANOVA followed by Scheffe's test (physiology), or

+/+ +/-

285 - '

BDNF probe

185 -

285 -

NT4 probe

185 -

GAPDH

FIG. 1. Northern blot with 40 ].Jg of total cortex RNA from wild­type and bdnf"l- mice was sequentially hybridized with bdnf, nt4. and GAPDH cDNA probes. Two bdnf mRNA bands were readily detected at the expected size. In contrast, nt4 mRNA signals were undetectable, even with prolonged autoradiography. GAPDH hybridization showed equal amounts of RNA samples on the blot.

bdnf OR nt4 GENE DELETION IN ISCHEMIC BRAIN DAMAGE 141

TABLE 1. Physiologic parameters in nt4-/-, bdnf+/-, and wild-type control mice (129/S�a)

Parameter wt nt4-1- bdnrl-

MABP (mm Hg) Before 99 ± 13 94 ± 14 109±3 During (0-29 min) 107 ± 10 108 ± 7 107 ± IS During (30-60 min) 102±9 101 ± 15 96±22 After 104 ± 3 103 ± 13 118 ± 13

rCBF (%) Before 100 ± 0 100±0 100±0 During IS ± 5 15 ± 9 18 ± 8 After 96± 12 96 ± II 90± 8

pH Before 7.33 ± 0.03 7.36 ± 0.02 7.36 ± 0.01 After 7.33 ± 0.06 7.35 ± 0.05 7.30 ± 0.05

Paco2 (mm Hg) Before 38 ± 5 41 ± 6 38 ± 3 After 40±6 42 ± 7 42±9

Pao2 (mm Hg) Before 150 ± 26 ISS ± 27 152 ± 14 After 143 ± 35 147 ± 26 101 ± 25

CT eC) During 36.8 ± 0.3 36.7 ± 0.3 36.8 ± 0.3

Weight (g) 21.9 ± 4.2 21.6 ± 4.6 22.3 ± 4.1

Animals were subjected to I hour of filamentous middle cerebral artery occlusion followed by reperfusion. Mean arterial blood pressure (in mm Hg; MABP) and regional cerebral blood flow (in % of baseline; rCBF) were measured before ischemia (baseline), during ischemia, and after 30 minutes reperfusion (Huang et aI., 1994; Endres et aI., 1997). Fifty microliters of blood were withdrawn twice, before ischemia and directly before withdrawal of the filament for blood gas determination (pH, Pao2, Paco2). Animals were weighed before onset of the experi­ment (body weight in grams). Rectal (core) temperature (CT in 0c) was controlled and kept constant by means of a feedback temperature­control unit (Endres et aI., 1998b). No statistical significant differences were found between groups (n = 4 animals per group; mean ± SD).

Mann-Whitney rank sum U test (neurological deficits), P val­ues smaller than 0.05 were considered statistically significant.

RESULTS

Bdnf and nt4 mRNA expression in the adult mouse

cerebral cortex

Northern blot analysis indicated that bdnf messenger RNA (mRNA) was more abundant than nt4 mRNA in the cerebral cortex (Fig. 1), in accordance with a previ­ous report for adult rats (Timmusk et a!. , 1993). Bdnfl­mice expressed reduced (50%) bdnf mRNA levels in the brain (Fig. 1) (see also Ernfors et a!., 1994). As previ­ously demonstrated, nt4-1- mice lack nt4 mRNA expres­sion (Liu et a!. , 1995).

Physiologic parameters

As displayed in Table 1, there were no significant differences in systemic physiologic or regional cerebro­vascular parameters between wild-type, nt4-1-, or bdnf'­mice before, during, or after transient MCAO.

Cerebral infarcts after transient middle cerebral

artery occlusion/reperfusion

After transient ischemia (I-hour MCAO and 23-hour reperfusion), infarcts in ntrl- mice were significantly

bigger (67%) than in wild-type 129/SVjae mice (Fig. 2A). This difference was also significant when lesion volume was calculated after correcting for brain swelling (28.3 ± 16.2 mm3 versus 47.6 ± 15.5 mm3 in wild-type versus ntrl- mice, respectively, P < 0.01). Moreover, sensori­motor deficits were significantly worse in ntrl- mice after 24 hours, indicating that larger infarcts were ac­companied by diminished central nervous system func­tion (Ll ± 0.8 versus 2.2 ± 0.8 in wild-type versus ntrl-mice, respectively, P < 0.01).

Bdnfl- mice developed larger infarcts (92%) com­pared to those of controls when subjected to I-hour MCAOI 23-hour reperfusion (Fig. 2A). Differences were

A 100

80

Infarct Volume 60 (mm3) 40

20

0

B 160

140 120

100 Infarct Volume (mm3) 80

60

40

20

0

129/SV

WT (n=15)

129/SV

WT (n=19)

**

NT4-I-(n=14)

**

NT4-1-(n=13)

**

BDNF+I­(n=10)

FIG. 2. (A) Infarct size was 69% larger in animals lacking nt4 (NT4+I-) and 92% in animals lacking one bdnfallele (bdnf+I-) after transient (1-hour) occlusion of the middle cerebral artery followed by 23-hour reperfusion compared to wild-type (WT) controls. Dif­ferences were also significant when infarction volumes were cal­culated indirectly (see text), and bigger infarcts in the transgenic mice were accompanied by worse functional outcome (see text). (8) Infarct size was 21% larger in nt4 knockout mice (NT4-1-) after permanent occlusion of the middle cerebral artery for 24 hours compared to wild-type (WT) controls (pure 129/SV back­ground). Differences were also significant when infarct volume was calculated by an indirect method (see text). Mean ± SO. **P < 0.01.

J Cereh Blood Flow Metab, Vol. 20, No.1, 2000

142 M. ENDRES ET AL.

WT

- : ........

. , ... ' . ..... :

. .

NT4-/-

also significant when measured by the indirect method (28.3 ± 16.2 mm3 versus 52.8 ± 27.7 mm3 in wild-type versus bdnfl- mice, respectively, P < 0.05). Neurologic deficits were also significantly worse after 24 hours 0.1 ± 0.8 versus 2.0 ± 0.7 in wild-type versus bdnfl- mice, respectively, P < 0.05).

In preliminary histologic studies (n = 2 to 3 per group), the same trend was noted (see Fig. 3). Except for differences in infarct area, qualitative differences were not detected in overall histopathology. Nevertheless, in ischemic striatum from wild-type mice, normal appear­ing NeuN-positive nuclei were found among positive nuclei with a condensed staining pattern; all nuclei showed this abnormal pattern in nt4-1- and bdnfl- mice (Fig. 4).

Effects of permanent ischemia in nt4-1- mice

Lesions were significantly larger (21 %) compared to wild-type controls in nt4-1- mice subjected to permanent ischemia for 24 hours (Fig. 2B). Differences were also significant when measured with an indirect method (80.1 ± 10.8 mm3 versus 97 ± 17.3 mm3 in controls versus nt4-1-, respectively, P < 0.01, n = 19 and 13 animals).

DISCUSSION

We evaluated outcome after MCAO in transgenic ani­mals either deficient in nt4 gene expression or expressing reduced bdnf levels. We found that these mutant mice were less resistant compared to controls, indicating that nt4 and bdnf may protect from ischemic injury. Bigger infarcts were accompanied by worse neurologic scores in both mutant strains, indicating that the increases in in­farct volume corresponded to poorer outcome in sen­sorimotor function at 24 hours. A more rapidly evolving

J Cereb Blood Flow Metab. Vol. 20. No.1, 2000

. . .-� .

. '- . . .. ,'H � , .. I!!>' •

f...,. : :: ..

'l��(

BDNF+/-

FIG. 3. Hematoxylin and eosin stain­ing of coronal sections representative of wild-type, bdnF1-, and nt4-1- mice after 1-hour middle cerebral artery oc­clusion and 23-hour reperfusion. The infarcted area (outlined with white dotted line) involves the striatum in all three animals. There is limited cortical infarction in the Wild-type (WT) whereas there is extensive cortical in­volvement in animals lacking one al­lele of bdnf (BDNF+1-) or both alleles of nt4 (NT4-1-).

lesion was also detected in both mutant strains by NeuN immunohistochemistry. In the striatum, nuclei were con­densed and irregular, whereas in wild-type mice, normal rounded-appearing nuclei were frequent (Fig. 4). The enhanced susceptibility to ischemia could not be ex­plained by differences in cardio- or cerebrovascular pa­rameters, Interestingly, absolute infarct volumes were larger in bdnfl- compared to nt4-1- mice (Fig. 2A), in­dicating that the loss of even one bdnf allele may be functionally more relevant than complete loss of nt4 gene expression. The percentage of increase in infarct size in ntrl- compared to control mice was less pro­nounced after permanent MeAO (Fig. 2). This may mean that the neuroprotective effects depend on the se­verity of the insult as has been suggested by others (Lindvall et aI. , 1994) or, alternatively, may simply re­flect a ceiling effect in the permanent model. Taken to­gether, we infer that nt4 and bdnf confer resistance to brain ischemia when expressed normally, We cannot ex­clude the possibility that differences in brain develop­ment in nt4 and bdnf mutants contribute to the differ­ences in stroke outcome, although gross morphologic appearance of the brain is normal in both mutants.

bdnf, nt4, and trkB, their common receptor, are abun­dantly expressed in the central nervous system especially in cortex and hippocampal sub sectors (Kokaia et aI., 1993; Timmusk et aI. , 1993; Yan et aI. , 1997; Friedman et aI. , 1998). NT4 and BDNF share a 54% amino-acid identity, and both bind with high affinity to TrkB (lp et aI. , 1992). We found that total brain bdnf mRNA levels were more abundant than nt4 mRNA which agrees with the literature (Fig. 1). Although not detectable by analy­sis of total RNA (Northern blot), widespread nt4 mRNA expression was demonstrated in the brain by RNAase

bdnf OR nt4 GENE DELETION IN ISCHEMIC BRAIN DAMAGE 143

., "1 .... ,.

f\ .. 1- .t'

�

�, .. ... If!" 0('-

,> ., ". "')", •

• !�.,. �

"'� ... �

.. fI C •

.. ..., to

- ! "\ b • f)

.. ", .- '"

• /I . .' ..... �

" �f " , " is II .:,.. ,

'\ • ., . .. . " II ... '"

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FIG. 4. NeuN immunohistochemical staining within striatum. Nonischemic striatal neurons exhibit rounded nuclei (A). After 1-hour middle cerebral artery occlusion and 23-hour reperfusion, NeurN-positive nuclei from nt4-1- (C) and bdnf"l- mice (0) were irregular and condensed. This change was significantly less prominent in wild-type mice (8) (arrows indicate more normal straining pattern). Scale bar is 20 �rn.

protection assay and polyA+ RNA Northern blotting, as was NT4 protein (Ip et aI, 1992; Timmusk et aI. , 1993; Friedman et aI. , 1998).

Indirect evidence supports an endogenous neuropro­tective role for trkB and its receptor agonists during brain ischemia. After both focal and global cerebral ischemia, neuronal upregu1ation of trkB (but not trkA or trkC) and bdnfmRNA (in contrast to nt3) was demonstrated (Lind­vall et aI. , 1992; Comelli et aI. , 1993; Merlio et aI. , 1993; Takeda et aI., 1993; Kokaia et aI. , 1995, 1996; Fried­mann et aI. , 1998; Narumiya et aI. , 1998). TrkB and bdnf mRNA expressing neurons are typically located in the penumbral zone (focal ischemia) or CA3 subsector of the hippocampus (global ischemia). Neurons, however, that are likely to die, (e.g. , in the ischemic core [focal isch­emia] or in the CAl hippocampal subsector [global isch­emia]) do not show enhanced gene expression (Takeda et aI., 1993; Kokaia et aI., 1996). However, neurotrophins may not always protect ischemic tissue. For example, bdnf may enhance excitatory synaptic transmission which could augment excitotoxic damage and increase the probability of seizures (Knipper et aI. , 1993; Lohof et aI., 1993; Kang and Schuman, 1995). Moreover, trun­cated TrkB isoforms may block bdnfsignaling (Knusel et aI., 1994; Eide et aI. , 1996). In contrast to the adult, expression of truncated TrkB receptors is low in the neo­natal period which may explain the more consistent pro­tective effects of BDNF in neonatal in contrast to adult models (Cheng et aI. , 1997).

The benefits of exogenous administration of several neurotrophins have been reported previously in focal and

global ischemia as well as in other brain insults (Beck et aI. , 1994; Tsukahara aI. , 1994; Chan et aI. , 1996; Alexi et aI. , 1997; Cheng et aI. , 1997; Schlibitz et aI. , 1997). For example, exogenous bdnf delivered intracerebroventricu­larly decreases lesion size after permanent focal (2.1 /-Lg/d for 8 days, beginning 24 hours before ischemia) (Schlibitz et aI. , 1997) and global cerebral ischemia (0.013 /-Lgih for 7 days, beginning immediately before ischemia) (Beck et aI, 1994) in rats. Similarly, intrace­rebroventricular administration of nt4 1 day before and immediately after occlusion confers protection in a rat permanent MCAO model. Infarcts were reduced by 34% 1 day after ischemia; however, infarction volume was unaltered 4 to 7 days after ischemia (2 x 0.54 /-Lg) (Chan et aI. , 1996).

In summary, our findings are consistent with the con­clusion that endogenous bdnf and nt4 are neuroprotective during MCAO in adult mice and underscore the potential importance of the trkB receptor in conferring resistance to ischemic injury.

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