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(Stroke. 2000;31:2707.)
© 2000 American Heart Association, Inc.

Original Contributions

Differences in Vulnerability to Permanent Focal Cerebral Ischemia Among 3 Common Mouse Strains

Arshad Majid, MB, ChB, MRCP(UK); Yong Y. He, MD; Jeffrey M. Gidday, PhD; Stuart S. Kaplan, MD; Ernesto R. Gonzales, BSN; T. S. Park, MD; Joseph D. Fenstermacher, PhD; Ling Wei, MD; Dennis W. Choi, MD, PhD Chung Y. Hsu, MD, PhD

From the Departments of Neurology and Neurosurgery and Center for the Study of Nervous System Injury, Washington University School of Medicine, St Louis, Mo, and Department of Anesthesiology, Henry Ford Hospital, Detroit, Mich (J.D.F.).

Correspondence to Chung Y. Hsu, MD, PhD, Department of Neurology, Box 8111, 660 S Euclid Ave, St Louis, MO 63110. E-mail hsuc{at}neuro.wustl.edu

	Abstract

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Background and Purpose—Genetically engineered mice are used to study the role of single genes in cerebral ischemia, but inherent, strain-dependent differences in neuronal vulnerability may affect experimental end points. To examine this possibility, tissue injury resulting from focal ischemia and its relationship to cerebral hemodynamics were determined in 3 common mutant mouse strains.

Methods—Permanent middle cerebral artery ligation was performed in male C57BL/6J, Balb/C, and 129X1/SvJ mice. Mean arterial blood pressure, blood gases, basal and postischemic cortical blood flow ([¹⁴C]iodoantipyrine autoradiography and laser-Doppler flowmetry), posterior communicating artery patency, and infarct size were determined.

Results—Basal cortical blood flow did not differ among strains. Ten minutes after middle cerebral artery ligation, relative red cell flow in the ischemic cortex was 6% to 7% of preischemic flow in every strain. Despite similar hemodynamics, cortical infarcts in Balb/C mice were 3-fold larger than those in 129X1/SvJ and C57BL/6J mice; infarct size in the latter 2 strains was not significantly different. The posterior communicating artery was either poorly developed or absent in >90% of the Balb/C and C57BL/6J but in <50% of the 129X1/SvJ mice.

Conclusions—The extent of ischemic injury differed markedly between the 3 strains. The presence and patency of posterior communicating arteries, although variable among strains, did not affect preischemic or postischemic cortical blood flow or bear any relationship to ischemic injury. Therefore, intrinsic factors, other than hemodynamic variability, may contribute to the differences in ischemic vulnerability among strains. These findings underscore the importance of selecting genetically matched wild-type controls.

Key Words: circle of Willis • genetic engineering • middle cerebral artery • mutation • posterior communicating artery • transgenics • mice

	Introduction

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The use of genetically engineered mice to study the function of single genes in cerebral ischemia has increased considerably in recent years. To date, the effects on stroke outcome of the overexpression of genes for CuZn superoxide dismutase,¹ basic fibroblast growth factor,² P-selectin,³ glutathione peroxidase,⁴ -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors,⁵ and amyloid precursor protein⁶ have been reported. With respect to underexpression, cerebral ischemic injury has been studied in animals deficient in the genes for neuronal,^{7 8} endothelial,⁹ and inducible nitric oxide synthase,¹⁰ metallothioneins,¹¹ interleukin-1ß,¹² leukocyte adhesion molecules,^{13 14} poly(ADP-ribose) polymerase,¹⁵ NADPH oxidase,¹⁶ tissue plasminogen activator,¹⁷ adenosine (A2) receptor,¹⁸ neurotrophins,¹⁹ and CuZn and mitochondrial manganese superoxide dismutase.^{20 21}

Since embryonic stem cells with inserted or deleted genes are easily derived from 129/Sv mice, most genetically engineered mice have 1 parent of the 129/Sv strain.²² The stem cells are then typically implanted into blastocysts, and the resulting chimeric mice carrying the manipulated gene are subsequently bred with other murine strains. For the latter, the C57BL/6 strain is usually chosen because they are easier to breed and are less susceptible to a number of diseases, but Balb/C and other murine strains have also been used.^{22 23} In addition, the extent of gene mixing can vary further depending on which generation of back-crossed mice is used for experimentation. Accordingly, although transgenic mice derived from a single parent strain have been generated,¹⁷ most mutants are derived from parents of 2 different strains and have a mixed genetic background. The resultant genetic profile of animals derived from such matings can influence the vulnerability or resistance to cerebral ischemia relative to the respective parent strain, one of which is often used for the wild-type control.^{7 8 12 17}

As indicated above, 129/Sv, C57BL/6, and Balb/C mice are common parent strains used in generating mutants. Variations in vulnerability to global forebrain ischemia among these strains of mice have been reported and ascribed to differences in the presence and relative size of the posterior communicating arteries (PComAs), which connect the anterior and posterior circulations and function as collateral vessels.^{24 25 26 27 28} With respect to focal ischemia, neurological outcome differences among strains have been published for the mouse intraluminal filament model of temporary middle cerebral artery (MCA) occlusion. For instance, larger infarcts have been found in C57BL/6 mice than in 129/Sv mice after focal transient ischemia.²⁹ The dependence of stroke outcome on murine strain after permanent MCA occlusion with the intraluminal filament technique is, however, unclear, especially with respect to anatomic differences in the PComA.^{7 29 30}

Highly reproducible cortical lesions are obtained by ligating the MCA directly after temporal craniotomy.^{6 10 20 31 32} This method interrupts the flow of blood through the MCA with more certainty than an intraluminally advanced filament, does not affect the anterior and posterior cerebral arteries, minimizes collateral contributions by the PComA, and yields less variable results.³³ To date, interstrain differences in infarct size resulting from cerebral ischemia induced by direct, permanent MCA ligation have not been reported. We hypothesized that there might be differences in vulnerability with the direct ligation model of permanent focal ischemia among the 3 mouse strains most commonly used in genetic engineering and tested the dependence of these "differences" on basal blood flow, the extent of blood flow reduction after MCA ligation, and anatomic differences at the level of the PComA.

	Materials and Methods

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In this study, 12±2-week-old Balb/C (n=49; from Charles River; Wilmington, Mass), 129X1/SvJ (n=45; former designation 129/SvJ), and C57BL/6J (n=65; both from Jackson Labs; Bar Harbor, Maine) mice were used. All procedures were approved by our institutional Animal Studies Committee and were in accordance with the Public Health Service guide for the care and use of laboratory animals, US Department of Agriculture regulations, and the guidelines of the American Veterinary Medical Association Panel on Euthanasia. Mice were allowed free access to water and chow until surgery.

Surgical Preparation
Focal cerebral ischemia was induced by direct occlusion of the MCA as detailed in previous publications,³⁴ with the following modifications. Briefly, the mice were anesthetized with ketamine (100 mg/kg) and xylazine (5 mg/kg), and the right MCA was exposed by a 0.5-cm vertical skin incision midway between the right eye and ear. After the temporalis muscle was split, a 2-mm burr hole was drilled at the junction of the zygomatic arch and the squamous bone. While visualizing with an operating microscope, we ligated the right MCA distal to the lenticulostriate branches with an 11-0 suture. Complete interruption of blood flow at the MCA occlusion site was confirmed by microscopic inspection and laser-Doppler flowmetry (see below). The right femoral artery was cannulated in some of the animals (n=5 in each strain) for monitoring arterial blood pressure and for obtaining blood samples for arterial blood gases. Blood pressure was monitored with a Digi-Med blood pressure analyzer (Micro-Med, Inc). Arterial blood gases were determined before ischemia and at 30 minutes after the onset of ischemia with a blood gas analyzer (model 238, Ciba Corning). Rectal temperature was recorded and maintained at 37.0±0.5°C before and for 1 hour after MCA ligation via an electronic temperature controller (Versa-Therm 2156, Cole-Parmer) linked to a heating lamp and homeothermic blanket control unit (Harvard Apparatus). After recovery from anesthesia, animals were allowed free access to food and water. All mice were housed in an air-ventilated room with ambient temperature set at 24±0.5°C for the ensuing 24 hours.

[¹⁴C]Iodoantipyrine Autoradiography
Four mice from each of the 3 strains were subjected to baseline (nonischemic) cortical blood flow measurement by means of iodoantipyrine autoradiography. Mice were anesthetized with a mixture of 1.5% halothane, 69% nitrous oxide, and 29.5% oxygen. Under the operating microscope, the femoral artery and femoral vein were catheterized on both sides of the animal with polyethylene tubing (PE-10; 3.0 cm long). The wound was infiltrated with lidocaine-HCl and closed with sutures. Body temperature was monitored and maintained at 37.0°C to 37.5°C with a heat lamp. Arterial blood pressure was continuously recorded, and arterial blood samples were taken for blood gas assays before the start of the actual measurement of flow. The measurement of cortical cerebral blood flow followed the procedures described by Jay et al³⁵ and Wei et al,³⁶ with some modifications. In brief, 5 to 10 µCi of [¹⁴C]iodoantipyrine (American Radiochemical) was infused into 1 femoral vein for 20 seconds; 6 well-timed blood samples were collected on preweighed pieces of filter paper over this period. At 20 seconds, the mice were decapitated. The brains were removed from the severed heads and frozen in 2-methylbutane cooled to -45°C within 30 seconds of decapitation. Frozen brains were stored at -80°C until the time of sectioning. ¹⁴C radioactivity was determined in the reweighed samples of blood by liquid scintillation counting. Tissue radioactivity was assayed by quantitative autoradiography in the same part of the cortical field of the MCA in all 3 strains. Coronal sections (20 µm thick) were serially cut in a cryostat set at -17°C, starting at the level of the area postrema and ending at the rostral end of the caudate putamen. These sections were placed in x-ray cassettes along with an appropriate set of standards and a sheet of x-ray film (BRS Kodak). Commercial standards were used for ¹⁴C quantification (American Radiolabeled Chemicals). The exposure period of the sections and standards was 7 to 9 days. The optical densities of the brain images and of the standards were measured on the autoradiograms with an MCID image analysis system (Imaging Research Inc). Cortical cerebral blood flow was determined from the blood and tissue radioactivities and the equation of the method.^{35 36}

Laser Doppler Flowmetry
Relative red cell flow in the core of the ischemic territory (lateral parietal cortex) was measured in 9 to 12 mice in each strain with a laser-Doppler probe (model 403A, Transonic Systems, Inc). The location for the cortical flow measurement was 2 mm posterior and 6 mm lateral to the coronal suture and distal (more superior) to the MCA ligation site. The tip of the micromanipulator-mounted laser-Doppler probe was placed against an intact dura through a small craniotomy made at this location, resulting in flow measurements directly above the core of the ischemic MCA territory.^{3 7 17 30} Several successive measurements were obtained before ligation and averaged to obtain a representative baseline value for red cell flow in the lateral cortex. Repeated measurements were then obtained again 10 minutes after MCA ligation. Changes in red cell flux were normalized to preischemic levels.

Morphometric Analysis of Infarct Volume
Twenty-four hours after permanent MCA ligation, animals were killed with an overdose of pentobarbital (100 mg/kg IP). Immediately thereafter, blood was washed out of the circulation by saline infused through the heart. The brains were removed and sliced into 2-mm coronal sections with a matrix (Harvard Bioscience). The brain sections were incubated in normal saline containing 2% triphenyltetrazolium chloride (TTC) (Sigma) at 37°C for 20 minutes and subsequently stored in 10% phosphate-buffered formalin. The total cross-sectional area and the unstained portion of each coronal slice were determined with an image analyzer (DUMAS, Drexel University). The volume of the infarct was calculated by measuring infarct areas on the separate slices, multiplying these areas by slice thickness, and summing all slices; the "indirect" morphometric method³⁷ was used to correct for edematous swelling. Incidentally, infarct volumes estimated by TTC staining and the indirect method agree with those determined from hematoxylin and eosin–stained histologies.³⁸

Carbon Black Labeling of Cerebral Arteries
In deeply anesthetized mice, carbon black (in an equal volume of 20% gelatin in distilled water) was perfused through the heart and into the vascular system of Balb/C (n=13), C57BL/6J (n=22), and 129X1/SvJ (n=15) mice under baseline (nonischemic) conditions. The animals were then killed with an overdose of pentobarbital. Brains were carefully removed, and the circle of Willis was examined under a dissecting microscope. The development of left and right PComAs was scored individually as follows: 0, absent; 1, present but poorly developed (hypoplastic); and 2, well formed. A single PComA development score was calculated for each animal by averaging the left and right scores.

Statistical Analyses
Intra-animal changes in physiological variables were assessed by paired t tests. Intrastrain and interstrain differences were assessed by 1-way ANOVA followed by post hoc Tukey tests. Infarct, cerebral blood flow, and red cell flow data are expressed as mean±SEM and were analyzed by ANOVA followed by post hoc Tukey tests. The PComA data were analyzed by nonparametric 1-way ANOVA on ranks (Kruskal-Wallis). For all tests, P<0.05 was considered significant.

	Results

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Before ligation, mean arterial blood pressure and arterial blood gases were similar among the 3 strains and were in the range reported by others for nonventilated mice (Table).^{22 28} After permanent occlusion of the MCA, these physiological variables remained unchanged in the 3 strains. Rectal temperature measurements at 6, 12, and 24 hours after surgery were not different among the groups.

View this table: [in this window] [in a new window]   Table 1. Mean Arterial Blood Pressure and Arterial Blood Gas Values in the 3 Mouse Strains Studied Before and 30 Minutes After Permanent Ligation of the MCA

In control mice, blood flow assessed by [¹⁴C]iodoantipyrine autoradiography in the lateral cortex (the MCA territory) was not significantly different among the 3 strains (Figure 1). Ten minutes after MCA ligation, the reductions in red cell flow (laser-Doppler flowmetry) in the ischemic lateral cortex relative to preischemic values were virtually identical in the Balb/C, C57BL/6J, and 129X1/SvJ mice (Figure 2). These data plus the control iodoantipyrine–quantitative autoradiography results (Figure 1) indicate that this model of MCA occlusion yields red cell flows in the cortex that are 6% to 7% of control and similar in all 3 strains of mice.

View larger version (58K): [in this window] [in a new window]   Figure 1. Mean (±SEM) rate of local blood flow to the parietal cortex (lCBF) in anesthetized, resting mice from the 3 strains. The measurements were made with the use of the quantitative autoradiographic–iodoantipyrine technique (n=4 mice per strain).

View larger version (45K): [in this window] [in a new window]   Figure 2. Percent reduction in red cell flow measured by laser-Doppler flowmetry in the core of the MCA territory 10 minutes after MCA ligation. The percent reduction in red cell flow was obtained by dividing the ischemic by the preischemic values. The differences in percent flow reduction between strains were not significant. (n=9 to 12 mice per strain).

The volumes of the TTC-demarcated lesions after 24 hours of occlusion differed among these mouse strains (Figures 3 and 4). The cortical lesions in Balb/C mice were 2.9 times larger than those in C57BL/6J mice (P<0.05) and 3.7 times larger than those in 129X1/SvJ mice (P<0.01). The infarct volumes were not significantly different in the C57BL/6 and 129X1/SvJ mice.

View larger version (86K): [in this window] [in a new window]   Figure 3. Representative coronal sections of TTC-stained brains from the 3 mouse strains 24 hours after permanent MCA ligation. The anterior faces of the coronal sections are shown. The right or infarcted side therefore appears on the left side of the image. The size of the TTC-demarcated lesion is much larger in Balb/C than in C57BL/6J and 129X1/SvJ mice. The lighter areas outside of the lesion on the ischemic hemisphere and on the nonischemic hemisphere of some sections from Balb/C and 129X1/SvJ mice are artifactual reflections produced by photographing wet sections.

View larger version (42K): [in this window] [in a new window]   Figure 4. Mean (±SEM) infarct volumes in the 3 strains of mice based on TTC staining 24 hours after direct permanent ligation of the MCA. The differences in infarct volumes were significant between Balb/C mice (n=17) and the other 2 strains at either the *P<0.05 or **P<0.01 levels. Differences in infarct size between C57BL/6J (n=22) and 129X1/SvJ (n=16) mice were not significant.

Differences in PComA development were found among the strains (Figure 5). The mean PComA scores of the Balb/C and C57BL/6J mice were identical and significantly less than that of the 129X1/SvJ animals. In detail, PComAs were absent unilaterally or bilaterally in 46% of the Balb/C and 45% of the C57BL/6J mice and poorly developed (hypoplastic) in 46% and 48% of these 2 strains, respectively. Fully formed PComAs were found in only 8% of the Balb/C and 7% of the C57BL/6J mice. In 129X1/SvJ animals, however, 50% of these arteries were well formed, 27% were hypoplastic, and 23% were absent.

View larger version (34K): [in this window] [in a new window]   Figure 5. Mean (±SEM) score of PComA development in the 3 strains of mice based on carbon black labeling. PComA development and patency were significantly greater in 129X1/SvJ (n=15) than in C57BL/6J (n=22) and Balb/C (n=13) mice. *Difference from Balb/C or C57BL/6J is significant (P<0.05).

	Discussion

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Our results show that Balb/C mice are significantly more susceptible to damage resulting from permanent focal cerebral ischemia than C57BL/6J and 129X1/SvJ mice. The greater vulnerability of the Balb/C mice was not associated with a larger reduction in flow to the MCA territory, since control rates of blood flow were nearly identical in all 3 strains and the drops in relative red cell flow after direct arterial ligation were similar. Nor were vulnerability differences correlated with the extent of PComA development and size, which is not surprising for this direct MCA ligation model. Thus, the dissimilar tissue injury after permanent focal ischemia does not appear to be linked to interstrain cerebrovascular differences but may instead be related to intrinsic variations in neuronal and/or glial vulnerability to ischemia among the different strains.

Barone et al,²⁴ using both focal and global ischemia models, were the first to show differences in susceptibility to cerebral ischemia among 3 mouse strains. They found significant variations in PComA development and patency that appeared to correlate with injury. Red cell flow was estimated only in Balb/C mice during brief periods of ischemia; therefore, a linkage between blood flow and damage among the strains could not be established. With respect to C57BL/6J and 129X1/SvJ animals, our findings with direct ligation and those of others with intraluminal filament obstruction^{7 9} indicate that cortical infarcts resulting from permanent MCA closure involved approximately 7% of the hemisphere (Figure 4). Differing from our findings, Connolly et al³⁰ reported that the infarct volume after 24 hours of permanent MCA occlusion by intraluminal filament was approximately 2% of the hemisphere in 129X1/SvJ mice and one tenth of that occurring in C57BL/6J mice.

There are a number of possible causes of the apparent inconsistencies among these reports. Anesthetic agents importantly affect outcome in experimental stroke models because of central and peripheral alterations in blood pressure, hemodynamics, and vascular tone.^{25 28} The anesthetic regimen used by Connolly et al³⁰ and us was, however, identical. Cerebral ischemic injury also depends on brain temperature, but this was controlled and similar in both studies. The differences in ischemia models used (permanent vis-à-vis transient occlusion) undoubtedly contribute to the dissimilarity in the results. In the present study the MCA was permanently ligated under direct visualization through a lateral craniotomy; this model has also been used by other investigators.^{10 24 31} Although it produces smaller infarcts than those obtained by obstructing the origin of the MCA with an intraluminal filament, the technique is straightforward, and complete blood flow interruption distal to the ligation is ensured. In contrast to all the other models, the ligature exclusively shuts off blood flow in the distal MCA and does not directly affect flow in any other major artery.

Variable results are more likely to occur with the intraluminal filament model. Suture size and length, which depend on animal weight,^{30 39} and resin/silicone coating of the suture and its resultant final diameter^{7 17 23} differ among laboratories.³⁵ With the intraluminal suture model, residual blood flow around the occluding filament could vary among individuals, strains, and groups of animals, yielding dissimilar lesions. The placement of the filament certainly affects flow not only in the MCA but also in the internal carotid, anterior cerebral, and posterior cerebral arteries, and the extent to which this occurs might vary significantly among experiments and strains.

Differences in blood flow during ischemia may be the cause of the disparity in brain tissue damage among mouse strains. Local rates of blood flow need to be determined with accuracy and good spatial resolution to address this possibility. In general, blood flow has been measured in mouse studies by laser-Doppler flowmetry.^{9 10 24 29 30} This technique estimates red cell flow in a small block of tissue under the probe relative to a reference level determined either on the ipsilateral side before the system is perturbed or on the contralateral side during occlusion; the technique does not yield an absolute rate of blood flow. The variables in the estimation of relative flow include time and site of measurement, the portion of the ischemic areas covered by the measurement, and the reference level chosen.

With respect to time, relative reductions in blood red cell flow in lateral cortex at the primary site of ischemia measured 10 minutes after direct ligation were identical among the 3 strains in the present study, falling to approximately 6% to 7% of preischemic flow. Similar degrees of flow reduction in C57Bl/6 and 129/SvJ^{29 30} animals have been reported immediately after occlusion of the MCA by an intraluminal filament. When measured 24 hours after permanent MCA occlusion by an intraluminal filament, however, Connolly et al³⁰ found that relative red cell flows were lower in C57BL/6J than 129X1/SvJ mice (ie, 40% to 50% and 70% to 80% of contralateral, respectively), and infarct size at this time was greater in the more ischemic C57BL/6J animals. These results suggest that blood flow to the ischemic territory in the 24-hour period after intraluminal blockage of the MCA improved more in 129X1/SvJ than in C57BL/6J mice. This "improvement" in red cell flow could be either the result or the cause of the lesser degree of injury in 129X1/SvJ mice. Accordingly, repeated measurement of blood flow over the first several hours of ischemia and longer is certainly warranted.

As to the reference level of laser-Doppler flowmetry, Connolly et al³⁰ used the contralateral side as reference, but we used the preischemic ipsilateral side as reference. Regardless of the reference level selected, interstrain differences in relative red cell flow reductions have not been detected by laser-Doppler flowmetry immediately after MCA occlusion in either our study or the aforementioned studies.^{9 10 24 29 30} A complication with the reference approach is that the absolute rates of blood flow may differ among these mouse strains under control, preischemic conditions, a problem that has been addressed in several reports. The autoradiographic technique with [¹⁴C]iodoantipyrine (Figure 1) and the indicator fractionation technique with [¹⁴C]isopropyliodoamphetamine^{12 23} have been used to determine the absolute rate of whole blood—not red cell—flow. Our present findings with the former method and those of others using the latter tracer^{12 23} indicate that rate of basal blood flow to the parietal cortex is very similar in resting Balb/C, C57BL/6J, and 129X1/SvJ mice. The relative drops in red cell flux found by laser-Doppler flowmetry in the ischemic core imply comparable rates of absolute blood flow among these strains during the initial minutes after MCA occlusion and therefore do not explain the considerably larger infarct volume in Balb/C mice (Figure 4).

Laser Doppler flowmetry estimates average red cell flow velocity and relative red cell flow in a volume of tissue under the probe. In rats, this volume has been assumed to be approximately 1.0 mm³; it may be similar in mouse brain. Thus, a single laser-Doppler flow probe could measure net red cell flow in only a portion of the field of injury in the mouse brain and miss important differences among loci, strains, and conditions. The autoradiographic technique of measuring whole blood flow provides fine spatial resolution throughout the brain and also produces serial histologies for determining tissue injury. To establish in detail the dependence of ischemic brain injury on blood flow, autoradiography will have to be undertaken in a way that minimizes premortem and postmortem movement of the flow marker because of the small size of the mouse brain.³⁵ This matter is currently under investigation (L. Wei, MD, et al, unpublished data, 2000).

Poorly formed or hypoplastic PComAs may reduce collateral blood flow between the anterior and posterior circulation and have been suggested to partially account for differences in susceptibility to global ischemia among strains.^{24 25 26 27 28} Connolly et al³⁰ reported that both C57BL/6J and 129X1/SvJ mice had intact, patent PComAs, but we and others^{24 25 26} have found that these vessels in the C57BL/6J strain were poorly formed and virtually unlabeled with circulating carbon black. Further confusing this issue, the present and other reports^{26 27} suggest that the degree of PComA plasticity varies among individual mice of a single strain. For 129X1/SvJ and C57BL/6J mice, distinct interstrain differences in intracranial cerebrovascular anatomy but no differences in infarct size were found by us with the ligation model, but just the opposite was reported by Connolly et al³⁰ for the filament model. Perhaps the best that can be said is the following: (1) the rate of blood flow throughout the various parts of the ischemic territory, probably over some period of time, set the initial tissue injury; (2) these local flow rates plus those in the adjacent tissue need to be measured to determine the cause of the dissimilarity in stroke outcome among mouse strains; and (3) a correlation among local blood flow, development of the PComA, and ischemic tissue damage has not been clearly established.

The preceding arguments suggest that not only the rate of lowered blood flow but also certain endogenous factors, potentially inherent to a single strain, may be important in determining neuronal outcome after permanent focal ischemia. Strain-specific differences among neuronal and/or glial functions have been reported. For instance, in a model of kainate-induced sustained seizures, 129/Sv mice exhibited greater injury in the CA3 hippocampal subfield than C57BL/6J mice.⁴⁰ Other anatomic, biochemical, and behavioral characteristics are also known to vary among mouse strains.^{41 42}

Because mutant mice are often derived from homologous recombination in embryonic stem cells, mixed genetic backgrounds characterize many transgenic and knockout mice. Because of genetic linkage, the use of embryonic stem cells from 129/Sv to carry a targeted gene deletion into another genetic background (C57BL/6 or Balb/C) will also lead to the transfer of a number of 129/Sv genes flanking the mutation to the F2 animals.²² Using progenies from these mixed genetic backgrounds for the determination of the effects of specific gene products on stroke outcome may lead one to falsely conclude that differences in brain injury after focal cerebral ischemia between the wild type and the particular mutant is due to the disruption or insertion of that particular gene when in fact these differences might be inherent in the different strains. Back-crossing with a single parent strain for at least 12 generations and using wild-type littermates from heterozygous matings as controls will reduce the contribution from one of the parental genomes to 1%, which is approximately 300 genes.²² Nonetheless, the chance that some of the 300 genes introduced from 129/Sv mice will have influences on brain function and recovery from ischemia in the resulting mutant is not negligible. In some studies, both parent strains of the particular experimental mutant have been used as wild-type controls with no significant differences between parent-strain susceptibility to a particular ischemic injury.^{7 10 29} Since background genes flanking the mutation of interest may still, however, have an effect on the phenotype of the new host, studying the individual parent strains is not the best substitute for the use of littermate controls. Even better wild-type controls would be the use of littermates from parents that have been back-crossed to a single strain for at least 12 generations. In the future, the hypothesis that a particular targeted mutation is indeed truly responsible for the observed phenotype may be more reliably tested by determining the effect of replacing the gene or the products of the missing gene on the phenotypic end point of interest. It is important to note that there is a distinction between 129/Sv and 129/SvJ mice. Many previous publications did not use the J strain for the genetic background.

In summary, results obtained in the present study indicate that ischemic tissue injury in brain after permanent MCA occlusion is independent of the rate of red cell flow in the core of the lesion in 3 strains of mice. Similarly, differences in intracranial cerebrovascular anatomy do not affect infarct size in the direct ligation model. The findings do not, however, address the dependence of injury on blood flow in penumbral tissue, a rather difficult measurement to make, or on changes in blood flow over time after ligation of the MCA. They do strongly suggest that there are interstrain differences in neuronal and/or glial responses and vulnerability to permanent, direct focal ischemia among 3 mouse strains. The importance of using the appropriate wild-type control group in murine cerebral ischemia studies is implied in these results.

	Acknowledgments

 
This work was supported by National Institutes of Health grants NS25545, 28995, and 40525 (Dr Hsu), NS32636 (Dr Choi), NS21045 (Dr Park), NS37372 (Dr Wei), and NS23393 (Dr Fenstermacher). We are grateful for the excellent technical assistance from A.R. Shah, MS, Ronald Perez, and Shah-Hinan Ahmed, MD, in these studies.

Received April 24, 2000; revision received June 27, 2000; accepted July 18, 2000.

	References

Top Abstract Introduction Materials and Methods Results Discussion References Introduction  References

 
1. Kinouchi H, Epstein CJ, Mizui T, Carlson E, Chen SF, Chan PH. Attenuation of focal cerebral ischemic injury in transgenic mice overexpressing CuZn superoxide dismutase. Proc Natl Acad Sci U S A. 1991;88:11158–11162.[Abstract/Free Full Text]

2. MacMillan V, Judge D, Wiseman A, Settles D, Swain J, Davis J. Mice expressing a bovine basic fibroblast growth factor transgene in the brain show increased resistance to hypoxemic-ischemic cerebral damage. Stroke. 1993;24:1735–1739.[Abstract/Free Full Text]

3. Connolly ES Jr, Winfree CJ, Prestigiacomo CJ, Kim SC, Choudhri TF, Hoh BL, Naka Y, Solomon RA, Pinsky DJ. Exacerbation of cerebral injury in mice that express the P-selectin gene: identification of P-selectin blockade as a new target for the treatment of stroke. Circ Res. 1997;81:304–310.[Abstract/Free Full Text]

4. Weisbrot-Lefkowitz M, Reuhl K, Perry B, Chan PH, Inouye M, Mirochnitchenko O. Overexpression of human glutathione peroxidase protects transgenic mice against focal cerebral ischemia/reperfusion damage. Mol Brain Res. 1998;53:333–338.[Medline] [Order article via Infotrieve]

5. Le D, Das S, Wang YF, Yoshizawa T, Sasaki YF, Takasu M, Nemes A, Mendelsohn M, Dikkes P, Lipton SA, Nakanishi N. Enhanced neuronal death from focal ischemia in AMPA-receptor transgenic mice. Mol Brain Res. 1997;15:52:235–241.

6. Zhang F, Eckman C, Younkin S, Hsiao KK, Iadecola C. Increased susceptibility to ischemic brain damage in transgenic mice overexpressing the amyloid precursor protein. J Neurosci. 1997;17:7655–7661.[Abstract/Free Full Text]

7. Huang Z, Huang PL, Panahian N, Dalkara T, Fishman MC, Moskowitz MA. Effects of cerebral ischemia in mice deficient in neuronal nitric oxide synthase. Science. 1994;265:1883–1885.[Abstract/Free Full Text]

8. Ferriero DM, Holtzman DM, Black SM, Sheldon RA. Neonatal mice lacking neuronal nitric oxide synthase are less vulnerable to hypoxic-ischemic injury. Neurobiol Dis. 1996;3:64–71.[Medline] [Order article via Infotrieve]

9. Huang Z, Huang PL, Ma J, Meng W, Ayata C, Fishman MC, Moskowitz MA. Enlarged infarcts in endothelial nitric oxide synthase knockout mice are attenuated by nitro-L-arginine. J Cereb Blood Flow Metab. 1996;16:981–987.[Medline] [Order article via Infotrieve]

10. Iadecola C, Zhang F, Casey R, Nagayama M, Ross ME. Delayed reduction of ischemic brain injury and neurological deficits in mice lacking the inducible nitric oxide synthase gene. J Neurosci. 1998;1997:17:9157–9164.

11. Majid A, Bessho Y, He YY, Hsu CY, Choi DW. Influence of metallothioneins on brain cell vulnerability to ischemia and zinc toxicity. Neurology. 1998;50:A153. Abstract.

12. Schielke GP, Yang GY, Shivers BD, Betz AL. Reduced ischemic brain injury in interleukin-beta converting enzyme-deficient mice. J Cereb Blood Flow Metab. 1998;18:180–185.[Medline] [Order article via Infotrieve]

13. Soriano SG, Lipton SA, Wang YF, Xiao M, Springer TA, Gutierrez-Ramos JC, Hickey PR. Intercellular adhesion molecule-1-deficient mice are less susceptible to cerebral ischemia-reperfusion injury. Ann Neurol. 1996;39:618–624.[Medline] [Order article via Infotrieve]

14. Connolly ES Jr, Winfree CJ, Springer TA, Naka Y, Liao H, Yan SD, Stern DM, Solomon RA, Gutierrez-Ramos JC, Pinsky DJ. Cerebral protection in homozygous null ICAM-1 mice after middle cerebral artery occlusion: role of neutrophil adhesion in the pathogenesis of stroke. J Clin Invest. 1996;97:209–216.[Medline] [Order article via Infotrieve]

15. Endres M, Wang ZQ, Namura S, Waeber C, Moskowitz MA. Ischemic brain injury is mediated by the activation of poly (ADP-ribose)polymerase. J Cereb Blood Flow Metab. 1997;17:1143–1151.[Medline] [Order article via Infotrieve]

16. Walder CE, Green SP, Darbonne WC, Mathias J, Rae J, Dinauer MC, Curnutte JT, Thomas GR. Ischemic stroke injury is reduced in mice lacking a functional NADPH oxidase. Stroke. 1997;28:2252–2258.[Abstract/Free Full Text]

17. Wang YF, Tsirka SE, Strickland S, Stieg PE, Soriano SG, Lipton SA. Tissue plasminogen activator increases neuronal damage after focal cerebral ischemia in wild-type and tPA-deficient mice. Nature Med. 1998;4:228–231.[Medline] [Order article via Infotrieve]

18. Chen JF, Huang Z, Ma J, Zhu J, Moratalla R, Standaert D, Moskowitz MA, Fink JS, Schwarzschild MA. A(2A) adenosine receptor deficiency attenuates brain injury induced by transient focal ischemia in mice. J Neurosci. 1999;19:9192–9200.[Abstract/Free Full Text]

19. Endres M, Fan G, Hirt L, Fujii M, Matsushita K, Liu X, Jaenisch R, Moskowitz MA. Ischemic brain damage in mice after selectively modifying BDNF or NT4 gene expression. J Cereb Blood Flow Metab. 2000;20:139–144.[Medline] [Order article via Infotrieve]

20. Kondo T, Reaume AG, Huang TT, Carlson E, Murakami K, Chen SF, Hoffman EK, Scott RW, Epstein CJ, Chan PH. Reduction of CuZn-superoxide dismutase activity exacerbates neuronal cell injury and edema formation after transient focal cerebral ischemia. J Neurosci. 1997;17:4180–4189.[Abstract/Free Full Text]

21. Murakami K, Kondo T, Kawase M, Li Y, Sato S, Chen SF, Chan PH. Mitochondrial susceptibility to oxidative stress exacerbates cerebral infarction that follows permanent focal cerebral ischemia in mutant mice with manganese superoxide dismutase deficiency. J Neurosci. 1998;18:205–213.[Abstract/Free Full Text]

22. Gerlai R. Gene-targeting studies of mammalian behavior: is it the mutation or the background genotype? Trends Neurosci. 1996;19:177–181.[Medline] [Order article via Infotrieve]

23. Silva AJ, Stevens CF, Tonegawa S, Wang Y. Deficient hippocampal long-term potentiation in alpha-calcium-calmodulin kinase II mutant mice. Science. 1992;257:201–206.[Abstract/Free Full Text]

24. Barone FC, Knudsen DJ, Nelson AH, Feuerstein GZ, Willette RN. Mouse strain differences in susceptibility to cerebral ischemia are related to cerebral vascular anatomy. J Cereb Blood Flow Metab. 1993;13:683–692.[Medline] [Order article via Infotrieve]

25. Fujii M, Hara H, Meng W, Vonsattel J-P, Huang Z, Moskowitz MA. Strain-related differences in susceptibility to transient forebrain ischemia in SV-129 and C57Black/6 mice. Stroke. 1997;28:1805–1810.[Abstract/Free Full Text]

26. Yang G, Kitagawa K, Matsushita K, Macbuchi T, Yagita Y, Yanagihara T, Matsumoto M. C57BL/6 strain is most susceptible to cerebral ischemia following bilateral common carotid occlusion among seven mouse strains: selective neuronal death in the murine transient forebrain ischemia. Brain Res. 1997;752:209–218.[Medline] [Order article via Infotrieve]

27. Kitagawa K, Matsumoto M, Yang G, Mabuchi T, Yagita Y, Hori M, Yanagihara T. Cerebral ischemia after bilateral carotid artery occlusion and intraluminal suture occlusion in mice: evaluation of the patency of the posterior communicating artery. J Cereb Blood Flow Metab. 1998;18:570–579.[Medline] [Order article via Infotrieve]

28. Murakami K, Kondo T, Kawase M, Chan PH. The development of a new mouse model of global ischemia: focus on the relationships between ischemia duration, anesthesia, cerebral vasculature, and neuronal injury following global ischemia in mice. Brain Res. 1998;780:304–310.[Medline] [Order article via Infotrieve]

29. Hara H, Huang PL, Panahian N, Fishman MC, Moskowitz MA. Reduced brain edema and infarction in mice lacking neuronal isoform of nitric oxide synthase after transient MCA occlusion. J Cereb Blood Flow Metab. 1996;16:605–611.[Medline] [Order article via Infotrieve]

30. Connolly ES, Winfree CJ, Stern DM, Solomon RA, Pinsky DJ. Procedural and strain-related variables significantly affect outcome in a murine model of focal cerebral ischemia. Neurosurgery. 1996;38:523–532.[Medline] [Order article via Infotrieve]

31. Nawashiro H, Martin D, Hallenbeck JM. Inhibition of tumor necrosis factor and amelioration of brain infarction in mice. J Cereb Blood Flow Metab. 1997;17:229–232.[Medline] [Order article via Infotrieve]

32. Martinou J-C, Dubois-Dauphin M, Staple JK, Rodriguez I, Frankowski H, Missotten M, Albertini P, Talabot D, Catsicas S, Peitra C, Huarte J. Overexpression of BCL-2 in transgenic mice protects neurons from naturally occurring cell death and experimental ischemia. Neuron. 1994;13:1017–1030.[Medline] [Order article via Infotrieve]

33. Schmid-Elsaesser R, Zausinger S, Hungerhuber E, Baethmann A, Reulen H-J. A critical reevaluation of the intraluminal thread model of focal cerebral ischemia. Stroke. 1998;29:2162–2170.[Abstract/Free Full Text]

34. He YY, Hsu CY, Ezrin AM, Miller MS. Polyethylene glycol-conjugated superoxide dismutase in focal cerebral ischemia-reperfusion. Am J Physiol. 1993;265:H252–H256.[Abstract/Free Full Text]

35. Jay TM, Lucignani G, Crane AM, Sokoloff L. Measurement of local cerebral blood flow with [¹⁴C]iodoantipyrine in the mouse. J Cereb Blood Flow Metab. 1988;8:121–129.[Medline] [Order article via Infotrieve]

36. Wei L, Craven K, Erinjeri J, Liang GE, Bereczki D, Rovainen CM, Woolsey TA, Fenstermacher JD. Local cerebral blood flow during the first hour following acute ligation of multiple arterioles in rat whisker barrel cortex. Neurobiol Dis. 1998;5:142–50.[Medline] [Order article via Infotrieve]

37. Goldlust EJ, Paczynski RP, He YY, Hsu CY, Goldberg MP. Automated measurement of infarct size with scanned images of triphenyltetrazolium chloride–stained rat brains. Stroke. 1996;27:1657–1662.[Abstract/Free Full Text]

38. Lin TN, He YY, Wu G, Khan M, Hsu CY. Effect of brain edema on infarct volume in a focal cerebral ischemia model in rats. Stroke. 1993;24:117–121.[Abstract/Free Full Text]

39. Hata H, Mies G, Wiessner C, Fritze K, Hesselbarth D, Brinker G, Hossman K-A. A reproducible model of middle cerebral artery occlusion in mice: hemodynamic, biochemical, and magnetic resonance imaging. J Cereb Blood Flow Metab.. 1998;18:367–375.[Medline] [Order article via Infotrieve]

40. Schauwecker PE, Steward O. Genetic determinants of susceptibility to excitotoxic cell death: implications for gene targeting approaches. Proc Natl Acad Sci U S A. 1997;94:4103–4108.[Abstract/Free Full Text]

41. Lathe R. Mice, gene targeting and behaviour: more than just genetic background. Trends Neurosci. 1996;19:183–186.[Medline] [Order article via Infotrieve]

42. Lipp HP, Collins RL, Hausheer-Zarmakupi Z, Leisinger-Trigona MC, Crusio WE, Nosten-Bertrand M, Signore P, Schwegler H, Wolfer DP. Paw preference and intra/infrapyramidal mossy fibers in the hippocampus of the mouse. Behav Genet. 1996;26:379–390.[Medline] [Order article via Infotrieve]

Editorial Comment

Pak H. Chan, PhD, Guest Editor

Neurosurgical Laboratories Stanford University School of Medicine Palo Alto, California

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References Introduction  References

 
Transgenic and knockout mutant mice have been widely used in stroke studies since the first such study was reported approximately 10 years ago.^R1 The successful creation of several reliable, reproducible, and technically challenging focal and global cerebral ischemia models and the availability of many genetically modified mice contributed to the explosion in the use of these animals in stroke research.^{R2 R3}

To dissect out the complex cellular and molecular mechanisms of ischemic cell death and regeneration, it is reasonable and perhaps essential to use genetically modified mice. However, knockout mutant mice are usually generated with 2 or more mixed genetic backgrounds. Concerns have been raised by stroke researchers that these mixed genetic backgrounds might render the results difficult to interpret when studies are conducted to compare the knockout mice with wild-type mice.

In addition, different hemodynamic physiological and anatomic differences may exist in various strains of wild-type mice. Such questions were raised in the accompanying article by Majid and colleagues. In a carefully done study, these investigators studied and compared absolute regional cerebral blood flow, physiological variables, PComA patency, and infarct size after permanent MCA occlusion in C57BL/6J, Balb/C, and 129X1/SvJ mice. They found that the degree of infarct size in a mouse strain is independent of all the hemodynamic variables studied, and they proposed that intrinsic factors may contribute to differences in ischemic vulnerability among strains. A similar importance of genetic determinants has been observed in excitotoxic cell death.^R4 These findings provide a unique and important message and perspective for stroke researchers and neuroscientists that genetically matched wild-type control mice should be used with knockout mutant mice for stroke studies.

Received April 24, 2000; revision received June 27, 2000; accepted July 18, 2000.

	References

Top Abstract Introduction Materials and Methods Results Discussion References Introduction  References

 
1. Kinouchi H, Epstein CJ, Mizui T, Carlson E, Chen SF, Chan PH. Attenuation of focal cerebral ischemic injury in transgenic mice overexpressing CuZn superoxide dismutase. Proc Natl Acad Sci U S A. 1991;88:11158–11162.

2. Kamii H, Kinouchi H, Sharp FR, Chan PH. A model of transient focal cerebral ischemia in the mouse. In: Ohnishi ST, Ohnishi T, eds. Central Nervous System Trauma: Research Techniques. Boca Raton, Fla: CRC Press; 1995:139–146.

3. Murakami K, Kondo T, Kawase M, Chan PH. The development of a new mouse model of global ischemia: focus on the relationships between ischemia duration, anesthesia, cerebral vasculature, and neuronal injury following global ischemia in mice. Brain Res. 1998;780:304–310.

4. Schauwecker PE, Steward O. Genetic determinants of susceptibility to excitotoxic cell death: implications for gene targeting approaches. Proc Natl Acad Sci U S A. 1997;94:4103–4108.

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