(Stroke. 1997;28:639-645.)
© 1997 American Heart Association, Inc.
Articles |
Transgenic Rats as Models for Studying the Role of Ornithine Decarboxylase Expression in Permanent Middle Cerebral Artery Occlusion
From the Nuclear Magnetic Resonance Research Group (J.L., O.H.J.G., R.A.K.) and Animal Biotechnology Group (R.S., A.J., J.J., L.I.A.), A.I. Virtanen Institute, University of Kuopio (Finland).
Correspondence to Dr Risto A. Kauppinen, NMR Research Group, A.I. Virtanen Institute, University of Kuopio, PO Box 1627, FIN-70211 Kuopio, Finland. E-mail kauppine{at}messi.uku.fi.
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Abstract |
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Background and Purpose Cerebral ischemia causes activation of ornithine decarboxylase (ODC) gene and subsequent accumulation of putrescine, which might either directly or indirectly affect the outcome of cerebral infarct. We developed a transgenic rat overexpressing human ODC, which was used to explore the effect of abnormally high putrescine concentration in the brain on the infarct volume after permanent middle cerebral artery (MCA) occlusion.
Methods The transgenic rats were produced by the pronuclear injection technique with the use of cloned human ODC gene. The right MCA was permanently occluded through craniotomy. ODC activity and polyamines were assayed in the infarcted and contralateral hemispheres. MRI was used to quantify T2 relaxation time, apparent diffusion constant (ADC), and infarct volume, which was also determined by 2,3,5-triphenyltetrazolium chloride.
Results Permanent MCA occlusion resulted in extensive activation of ODC, which was approximately sevenfold greater than in syngenic animals at 20 hours after occlusion. Consequently, putrescine increased from approximately 10 and 230 pmol/mg to 160 and 410 pmol/mg in the infarcted hemisphere of syngenic and transgenic animals, respectively, but all the other polyamines were unchanged. This high putrescine in the transgenic rats did not influence infarct size evolution, as determined by MRI, T2, ADC, or the infarct volume by 2,3,5-triphenyltetrazolium chloride at 48 hours.
Conclusions Data from the ODC transgenic rat model show that the development of brain infarct after permanent MCA occlusion was not influenced by extensive levels of putrescine, indicating that this endogenous amine is not involved in maturation and spread of stroke lesion in vivo. Thus, it seems that ODC activation reflects an endogenous adaptation of neural cells to a noxious stimulus that does not directly influence lesion development.
Key Words: cerebral ischemia, focal • nuclear magnetic resonance • polyamines • rats, transgenic
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Pioneering studies of the use of transgene technology in cerebral stroke research were conducted in 1991 by Kinouchi and coworkers1 as mice overexpressing ZnCu superoxide dismutase were subjected to MCA occlusion. Now transgene methods are widely used to test the role of the overexpression of proteins that are putatively linked with given disease pathogenesis in a number of disease states.2 3 Transgene methods have been proposed to facilitate searching for plausible endogenous factors that may cause cellular pathology after noxious insults. For instance, the early work by Kinouchi et al1 demonstrated a neuroprotective effect of enhanced free radical removal and thus indicated strategies for drug design. Current transgenic animal models are most often mice, which imposes certain limits on their use as models for human disorders.
In this institute, several mouse lines have recently been generated overexpressing enzymes of polyamine metabolic pathways (for a review, see Reference 33 ). Of these animals, those with grossly elevated ODC activity in a number of organs have been subsequently tested for their sensitivity to develop cancer after exposure to ultraviolet light, low-frequency electromagnetic radiation, and carcinogens. The ODC mice show no increased rate of tumorigenesis in the skin (Reference 44 and T. Kumlin et al, unpublished data, 1997), however. These findings are at variance with a previous work on a transgenic mouse line with a different type of promoter in the gene construct.5 Given the number of documented actions of polyamines on brain electric functions,6 receptors and ion channels,7 8 9 10 and transmitter uptake and release,11 12 these animals are expected to also serve as valuable models for studies of the neurobiological role of polyamines. Early studies from our laboratory show that cerebral overexpression of ODC with grossly elevated putrescine affects some aspects of neurochemical13 and behavioral14 functions.
Time-dependent activation of ODC and putrescine accumulation after either global complete,15 16 incomplete,17 or focal18 brain ischemia have been implicated as causative factors of neuronal necrosis.9 Putative mechanisms of action by putrescine might involve the NMDA receptor9 or intracellular calcium homeostasis.19 On the other hand, there is evidence that does not support this hypothesis.18 20 Our previous studies of ODC transgenic mice show that grossly elevated cerebral putrescine does not influence histopathology in aged animals21 and that exposure of these animals to transient, incomplete forebrain ischemia does not affect the outcome of neuropathology in the hypoxia-vulnerable brain regions.17 These observations tend to support the idea that neither chronically nor acutely elevated putrescine per se is neurotoxic, thus favoring the aforementioned conclusions.
To further explore the role of severe putrescine accumulation in the brain, we generated transgenic rats that overexpress human ODC and exposed them to permanent occlusion of the MCA to study the possible contribution of altered polyamine profiles to the development of neuronal cell damage in vivo.
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Materials and Methods |
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Production of Transgenic Rats
The transgenic rats (Wistar strain) were produced by the standard pronuclear injection technique.22 The donor females weighing approximately 75 g were superovulated with intraperitoneal injections of 10 IU of pregnant mare's serum gonadotropin and 10 IU of human chorionic gonadotropin 3 days and 1 day, respectively, before the collection of the zygotes. After the injection of human chorionic gonadotropin, the females were mated with mature males. To maximize the number of recipients available, the phase of the estrous cycle of the recipient females was estimated with the use of vaginal smear preparations. The females in proestrus/early estrus were made pseudopregnant by mating them with vasectomized males. The zygotes microinjected with the intact human ODC gene23 were transferred into the oviducts of the recipient females immediately after microinjection. The detection of the transgenic rats was performed from tail biopsies by polymerase chain reaction as described by Halmekytö et al.23
Focal Ischemia
Eleven syngenic and 11 transgenic male Wistar rats were used in
the studies of permanent MCA occlusion induced as described by Chen et
al.24 Briefly, animals were anesthetized with 1% to 1.5%
halothane in a mixture of 70% N2O/30% O2. The
right MCA was exposed through a small craniotomy hole to the skull that
was drilled between eye and external auditory canal. The dura was
carefully pierced, and the MCA was elevated with a small surgical hook
and electrocoagulated. The hole was subsequently blocked with acrylic
repair material (Dentsply Ltd). Immediately after the MCA occlusion
procedure, the common carotid arteries were exposed, dissected free
from the surrounding connective tissue and the vagus nerve, and
temporarily occluded with miniature aneurysm clips for 60 minutes.
Rectal temperature was monitored throughout the entire procedure and
kept at 37.0±0.5°C with a heating blanket.
In another group of syngenic and transgenic animals, blood pH, Pco2, and Po2 were monitored during MCA occlusion. For the blood gas analysis, the femoral artery was separated from the vein and the nerve and cannulated, and 100-µL blood samples were collected before, 30 minutes after, and 60 minutes after occlusion. The samples were analyzed with Radiometer ABL-5 blood gas analyzer.
Determination of Cerebral Polyamines and ODC Activity
Brains of both syngenic and transgenic rats were removed 1 day
after focal ischemia. The infarcted and contralateral cerebral cortices
were separated from each other, striatum, and cerebellum and frozen
with liquid nitrogen. The cortices were then homogenized into twofold
volume of buffer containing 25 mmol/L Tris, 0.1 mmol/L EDTA,
and 1 mmol/L dithiothreitol (pH 7.4), and the activity of ODC was
assayed by a radiolabel method.25 For the polyamine
determination, 20 µL of the homogenate was extracted with 5%
sulfosalicylic acid, and putrescine, spermidine, and spermine were
assayed by a high-performance liquid chromatography
method.26
MRI Methods
MRI of rats was performed with the use of a microimaging console
(Surrey Medical Imaging) interfaced to a vertical 9.4-T Oxford
Instrument magnet (bore size, 89 mm) equipped with actively
shielded gradient coils (Magnex Scientific) 20 hours after exposure to
MCA occlusion. Animals anesthetized with halothane (1% in 70%
N2/30% O2) were fixed to a head holder and
positioned in the magnet bore in a standard orientation relative to
gradient coils. A birdcage resonator (diameter of 38 mm) was used
for signal transmission and reception. In all experiments the image
matrix size was 128x128, slice thickness was 0.5 mm (volume
determination) or 1.0 mm (T2 and ADC determination), and field of
view was 40 mm. Spin-echo multislice coronal pilot images (TR,
2000 ms; TE, 50 ms) were acquired for correct positioning of the
transversal sections recorded for infarct volume, brain water T2, and
ADC measurement. Infarct volume was determined from the multislice
transversal images (TR, 2000 ms; TE, 50 ms) with a 0.6-mm
center-to-center distance of slices by measuring the area of the bright
T2-enhanced region in each of the slices.
T2 relaxation times in the infarcted and normal contralateral cerebral cortex were calculated from the MRI located through the center of the infarct. Image intensities from the spin-echo sequence (TR, 2000 ms) with four different TE values (20, 40, 60, and 80 ms) were fitted to a monoexponential function to calculate T2.
ADC in the infarcted and normal cortex was measured with the use of a Stejskal-Tanner–type pulsed field, gradient spin-echo sequence27 with diffusion weighting along the phase-encoding direction. The diffusion gradient duration was 5 ms, and the diffusion time 48.5 ms. Four consecutive images with b values of 19.1, 76.6, 172, and 306 s/mm2 with TE of 60 ms were acquired to quantify ADC.
Determination of Infarct Volume by TTC
Animals were decapitated 48 hours after ischemia, and their
brains were cut into 1-mm slices with a McIlwain tissue chopper. Slices
were incubated in a 37°C medium containing 0.3 mol/L
K2HPO4 and 1.2% TTC for 30
minutes28 ; the volume of unstained infarct was determined
with the use of an image processing program on a personal computer.
Statistical Analysis
Unpaired Student's t test was used to compare
results from different animal groups, and paired Student's
t test was used when intraindividual data were analyzed.
Values indicated are mean±SEM.
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Results |
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Transgene Analysis and Phenotype of ODC Rats
Four transgenic founder rats were obtained with the use of the same intact human ODC construct as used for production of transgenic mice.23 Rats of the transgenic line UKUR6 exhibited elevated ODC activities in all tissues studied, most dramatically in testis, liver (data not shown), and brain, and were used in subsequent experiments with their syngenic littermates.
ODC transgenic rats have no abnormal visible features in their appearance, and their survival until 10 months did not differ from syngenic littermates. Male transgenic and syngenic rats weighed at the age of 6 to 7 weeks 178±5 and 183±1 g (n=4), respectively. The volumes of cerebrum and cerebellum as determined from T1-weighted (TR, 600 ms; TE, 20 ms) MR images did not differ between the two animal groups at the age of 7 weeks (data not shown). In these images with in-plane resolution of 270 µm, no obvious anatomic differences in the brains of the two animal groups were evident.
Consequences of MCA Occlusion
Blood gas analyses of a group of animals showed that pH,
Pco2, and Po2 were maintained close to
physiological levels during the insults in both syngenic and transgenic
rats (Table
). None of the animals died before planned
time of death.
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The infarct was detectable 20 hours after reperfusion in the
T2-weighted MR images in all 11 animals studied as a hyperintense area
(Fig 1). The infarction was present in the cerebral
cortex in all the animals and in 2 of 11 rats reached the striatum (one
from each group). Infarct volumes as determined from T2-weighted MRI
were not statistically different between the syngenic and transgenic
animals (Fig 2). The scatter of T2 hyperintensities was
greater among the transgenic than syngenic rats, and the two smallest
cortical lesions were determined in the transgenic animals.
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The T2 relaxation times were measured from the center of infarct and
unexposed contralateral cerebral cortex. In transgenic animals, T2 in
the infarcted and normal cortical brain tissue was 64±2 and 39±1 ms
(P<.01, paired Student's t test, n=6),
respectively. In syngenic rats, the respective relaxation times were
64±2 and 38±1 ms (P<.01, paired Student's t
test, n=5). T2 maps constructed on a pixel-to-pixel basis of syngenic
and transgenic animals (Fig 1
) show that there were no brain tissue
regions in the selected slice with abnormally high relaxation time
other than the infarct.
No regions in the slice through the center of the infarct other than
those closely corresponding to the T2 hyperintensity showed reduced
signal intensity in the diffusion-weighted images (data not shown). In
the contralateral cortex, ADC was
1.01±0.009x10-9 and
1.00±0.009x10-9 m2/s at 20 hours
after occlusion in syngenic and transgenic rats, respectively.
Volume-averaged ADC values in the infarct of
0.64±0.009x10-9 and
0.61±0.007x10-9 m2/s for
syngenic and transgenic rats, respectively, were indistinguishable (Fig 3).
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The infarct volumes 2 days after focal ischemia, as determined with TTC
staining, were not statistically different between syngenic
(50.7±10.7 mm3, n=6) and transgenic (70.3±6.3
mm3, n=5) animals (Fig 2).
ODC Activity and Polyamine Profiles
Cerebral cortex ODC activity was approximately 54-fold greater in
the transgenic rats than in their syngenic littermates before ischemic
exposure (Fig 4). ODC activities in the brain regions of
unexposed transgenic rats were as follows: frontal cortex, 23.5±0.9;
parietal cortex, 23.6±1.7; striatum, 25.5±2.1; hippocampus,
25.3±0.9; and cerebellum, 4.2±0.2 pmol CO2/mg wet wt per
hour (n=4). ODC in the brain cortex was significantly elevated by
2.5-fold in transgenic rats 20 hours after focal ischemia (Fig 4).
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As expected from the high constitutive ODC activity, the putrescine
concentration was approximately 7-fold greater in transgenic than in
syngenic animals (Fig 5). A massive accumulation
of cerebral putrescine occurred after ischemia in both types of animals
(Fig 5). In fact, putrescine in the transgenic rats was at the same
level as spermidine and spermine (Fig 5). The concentrations of higher
polyamines spermidine or spermine did not change after focal ischemia
in either animal group (Fig 5).
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Discussion |
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Both global and focal brain ischemia cause a number of neurochemical changes, of which activation of ODC and putrescine accumulation are among the most striking. It has been suggested that these factors might either alleviate or exacerbate brain infarction through direct or indirect mechanisms. Our data from the transgenic rat model show that neither (1) constitutively high ODC and putrescine nor (2) further short-lasting and extensive elevation of these biochemical factors affect the subsequent development of the ischemic stroke lesion and that (3) high ODC activity does not lead to spread of infarction beyond the region of MCA supply. It ought to be noted that there is a substantial induction of endogenous ODC with moderate putrescine accumulation in the infarcted cortex. It may be that the effect of transgene to these factors does not exacerbate the ischemic lesion and its volume. This idea is substantiated by recent reports that show that in transient focal ischemia in rat, inhibition of ODC by difluoromethylornithine reduces infarct volume29 and that in transient global ischemia in gerbil the inhibitor reduces reperfusion edema.30 The present data show that unphysiologically high ODC activity does not directly influence tissue outcome in permanent ischemia, however. Interestingly, Chan and coworkers31 have recently shown that in permanent MCA occlusion, overexpression of superoxide dismutase is not neuroprotective, unlike the situation previously reported for transient focal ischemia.
Compared with the multitude of transgenic mouse models in biomedical research, the number of similar rat models is scarce. This is partly due to the difficulty in handling and culture of rat embryos, although the transgene methodology is to a large extent the same for both mice and rats. For some experimental approaches rats are preferred over mice because of their larger size and/or special methods standardized for rats. For these reasons we produced the ODC transgenic rats, although similar transgenic mice were already in-house. The transgenic rats resembled closely the respective transgenic mice23 in that they overexpressed ODC in almost all tissues and consequently overaccumulated putrescine. Furthermore, there appeared to be no conversion of putrescine to the higher polyamines spermine and spermidine, a situation observed earlier in ODC transgenic mice.32 Here we have shown that even the massive accumulation of cerebral putrescine after ische-mia did not result in elevated concentrations of spermidine and spermine, indicating that there is strict regulation in the maintenance of the normal levels of the higher polyamines. The nature of the block between putrescine and the higher polyamines remains to be studied.
The focal ischemia model used results in a cortical infarction of approximately 50 to 70 mm3 in volume, which is almost exactly half of that reported with an intra-arterial thread model quantified by similar MRI methods.18 The major difference between the two models is that the latter one occludes the MCA and anterior communicating artery so that the striatal blood flow is sufficiently reduced to result in ischemic damage. The choice of occlusion model does not influence the conclusions of the present study with respect to the proposed mechanism of action of polyamines through the NMDA receptor in stroke, however. It has been shown that the effects of spermidine and spermine on binding of MK-801 are strong in caudate putamen and in layers 1 to 2 and 3 to 5 of cerebral cortex33 and that putrescine does not have a direct effect on this binding on either of the anatomic sites. In contrast, putrescine acts as a weak competitor of both spermidine and spermine for binding at the NMDA receptor.34 35 Thus, one would expect that the plausible effects of all the polyamines on stroke evolution and final volume that might be mediated through the NMDA receptor would be present in both cortical and striatal stroke. Ifenprodil, a drug that acts on the polyamine-binding site of the NMDA receptor, has been reported to reduce the cortical stroke lesion but not the striatal stroke lesion in rats.36 Similarly, in 1992 Sauer and coworkers18 showed that a competitive NMDA antagonist, d-(E)-2-amino-4-methyl-5-phosphono-3-pentanoic acid, reduces the cortical infarction but not the striatal lesion area. However, our data strongly suggest that the changes in the polyamine profiles that accompany focal brain ischemia have no direct influence on development of infarct.
We used both T2-weighted MRI and TTC to assess infarct volume in the cerebral cortex. It has been established that tissue edema as detected by T2-weighted MRI matches the histological infarction in the rat by 24 hours of ischemia.37 38 39 The infarct volumes by MRI at 24 hours and by TTC at 48 hours in syngenic animals were very similar, a fact that is consistent with a report by Knight et al.38 In the transgenic rats, a trend toward increase in stroke volume between 24 and 48 hours is seen, but since the scatter of infarct volumes was so large, no definitive conclusion of stroke size increase between the two time points can be drawn. It is possible that anatomic variation in the cerebral vasculature is responsible for the scatter in the infarct volumes rather than an effect of the transgene.
Brownian diffusion of brain water reduces within minutes after the onset of the ischemic energy failure40 41 as a consequence of cessation of two-directional water exchange between the extracellular and intracellular compartments. ADC is generally considered an index of cytotoxic edema within the first day of stroke, but during the maturation of the infarct over 2 to 3 days, ADC normalization closely reflects neuronal cell necrosis.38 We determined ADC, or more specifically the sum of Dxy, Dyy, and Dzy components of the diffusion tensor (see Reference 4242 for further details), at 20 hours after induction of focal ischemia, when cytotoxic edema is expected to be severe. The values for ADC obtained in normal brain tissue are in agreement with those reported for rat brain with similar MRI methods at 7 T43 44 but somewhat higher that those reported at lower field strength with large b values.39 The difference in the absolute ADC values between the present study and the literature might be due to the fact that we used relatively low diffusion weighting in our experimentation. Nevertheless, the change in ADC between ipsilateral and contralateral hemispheres we quantify in the rats agrees well with the majority of the other studies.38 39 40 43 45 Similar reduction of ADC in syngenic and transgenic animals as well as the close match between the volume of low ADC in the two animal groups shows that high ODC activity with putrescine accumulation does not affect cytotoxic edema in this model of stroke.
In conclusion, a transgenic rat line overexpressing human ODC was generated with high constitutive putrescine concentration in the brain. Development of brain infarct in vivo after permanent MCA occlusion was not influenced by extensive levels of putrescine, showing that this endogenous amine is not involved in maturation of stroke lesion in vivo.
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Selected Abbreviations and Acronyms |
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Acknowledgments |
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This study was supported by grants from the Academy of Finland and the Northern Savo Cultural Foundation. We are grateful to Dr Maria Halmekytö for fruitful discussions and Tuula Reponen for expert technical assistance.
Received September 25, 1996; revision received November 13, 1996; accepted November 26, 1996.
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