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(Stroke. 1996;27:498-503.)
© 1996 American Heart Association, Inc.

Articles

Gender Influences the Magnitude of the Inflammatory Response Within Embolic Cerebral Infarcts in Young Rats

Kewa Li, MD; Nancy Futrell, MD; J. Samuel Tovar; LiJuan C. Wang, MD; David Z. Wang DO Lonni R. Schultz, PhD

From the Departments of Neurology (K.L., N.F., L.C.W., D.Z.W.) and Biochemistry and Molecular Biology (N.F.), Medical College of Ohio, Toledo; Department of Biostatistics and Medical Epidemiology, Henry Ford Hospital, Detroit, Mich (L.R.S.); and Department of Neurology, Creighton University School of Medicine, Omaha, Neb (J.S.T.).

Correspondence to Nancy Futrell, MD, Department of Neurology, Medical College of Ohio, 3000 Arlington Ave, PO Box 10008, Toledo, OH 43614-0008.

	Abstract

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Background and Purpose The inflammatory response within cerebral infarcts may have an influence on tissue damage. Since old animals with an impaired immune response have decreased inflammation after experimental cerebral infarction, we postulated that female animals with an increased immune response will have an increased inflammatory response after cerebral infarction.

Methods Embolic cerebral infarcts were produced by photochemical irradiation of the right carotid artery in 12 female Fischer rats. The inflammatory response within 4-day-old infarcts was quantitated by histology with the use of computer-assisted image analysis and compared with that in 12 male rats from a previous series.

Results Severe infarcts had the most pronounced inflammatory response. Female rats had an increased inflammatory response in infarcts of all severity, which was statistically significant in severe cerebral infarcts even after adjustment for infarct size. Severe infarcts in males were significantly larger than those in females.

Conclusions Gender influences the outcome of embolic cerebral infarcts after photochemical damage to the carotid artery, both in terms of the magnitude of the inflammatory response and infarct size. There are numerous gender-related differences in neurochemicals, cytokine production, and drug metabolism that may influence tissue damage after stroke and responsiveness to therapeutic intervention. The preponderance of male animals in stroke research may produce results not applicable to female stroke patients. The use of female animals will be required to provide adequate models for the study of stroke in women.

Key Words: cerebral infarction • gender • immunology • inflammation • rats

	Introduction

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Gender bias in clinical research is a major issue in American medicine and politics. Because of the failure to include women in clinical trials,¹ there have been delays in determining effective treatment for women with various diseases. For example, gender-related differences occur in the response to platelet-inhibiting agents used for stroke prevention,² with recommendations for treatments in men being made more than a decade before those for women.^{3 4} Similarly, gender-related differences in morbidity and mortality from thrombolytic treatments have been described.⁵

Gender bias in clinical medicine may also influence the availability of clinical services offered to men and women with similar disease processes and similar disease severity. Gender differences in both diagnostic evaluations⁶ and major therapeutic interventions^{7 8} have been reported in patients with cardiac disease, with a suggestion that less aggressive diagnosis and treatment were provided to women.⁷ Gender bias can also be seen in the approach to animal studies of human disease. Most animal studies have been done on male animals, with results being generalized to females without validating this generalization.⁹ Stroke research is certainly an example. Animal research is done mainly on young male animals, with the results being generalized to men and women. The validity of this practice has never been tested. The discrepancy between the age and gender of the animals used in research compared with the age and gender of patients at risk for stroke could be one reason for the disappointing correlation between animal studies and clinical trials in stroke.^{10 11}

One measure of the validity of generalizing stroke experiments done on men to both genders could be tested by comparing the inflammatory response to stroke in male and female animals. Gender-related differences in immune function, which could alter the inflammatory response after stroke, are similar between species. Females have increased immune activity,¹² resulting in increased autoantibody production¹³ and increased rejection of transplanted tissue.¹⁴ The inflammatory response within cerebral infarcts¹⁵ may modulate acute tissue damage¹⁶ and repair,¹⁷ suggesting that gender-related differences in immune function could be an important factor in stroke.

Our work in old animals, which have decreased immune function,¹⁸ demonstrates a decrease in the inflammatory response after embolic stroke.¹⁹ We tested the hypothesis that the increased immune responses in female rats would lead to increased inflammation within cerebral infarcts. To determine whether studies on the inflammatory response to strokes in male animals can be generalized to females, we studied macrophage infiltration in embolic cerebral infarcts in male and female rats.

	Materials and Methods

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All procedures were approved by the Institutional Animal Care and Use Committee at the Medical College of Ohio, in accordance with the Guide for the Care and Use of Laboratory Animals (US Department of Health and Human Services, Public Health Service publication NIH 86-23, revised 1985). Embolic stroke was produced in 12 female Fischer rats, 2 months of age. Tissues from 12 male animals, also 2 months of age, from a previous series were used.¹⁹ The weight of the female rats ranged from 150 to 175 g; the weight of the male rats ranged from 225 to 280 g. Each rat was anesthetized with chloral hydrate 4.5% (12 mL/kg IP), which was supplemented as needed for all surgical procedures. Embolic stroke was produced by photochemical irradiation of the carotid artery.²⁰ Briefly, the right common carotid artery was surgically exposed, and two tandem segments, each 2 mm in length, were sequentially irradiated for 15 minutes with a helium-neon laser (632 nm, 200 mW/cm²) (model CR 90-5SMM, CR Laser) immediately after the injection of 5 mg/kg IV of hematoporphyrin derivative (Porphyrin Products) diluted to a total volume of 2.5 mL/kg.

All rats were killed 4 days later under intraperitoneal chloral hydrate anesthesia by transcardiac perfusion with 300 mL of saline followed by 300 mL of 10% neutral-buffered formalin at a pressure of 100 mm Hg controlled by a blood pressure manometer. Animals were decapitated, and the heads were immersed in the fixative. The brains were removed at least 2 hours later and placed in fixative. Twenty-four hours later the brains were cut into five 3-mm coronal pieces, processed, and embedded in paraffin. Four coronal sections (two per slide), 7 µm in thickness, were cut with a microtome at intervals of 1.5 mm through the brain. One slide was stained with hematoxylin and eosin and the other with glial fibrillary acidic protein (GFAP) immunoperoxidase with the use of the avidin-biotin (ABC) kit (Vector Scientific). Stained slides were evaluated with the use of a Nikon Microphot microscope interfaced with the MCID image analysis system (Imaging Research).

Infarct severity was graded as severe, moderate, or mild. Hematoxylin and eosin stain was used to assess neuronal damage. GFAP was used to identify astrocytes.²¹ Normal GFAP indicates little or no damage to astrocytes, with increased GFAP after mild tissue damage (reactive gliosis with increased synthesis of the GFAP).²² Loss of GFAP staining has been reported within severely damaged (infarcted) tissue.¹⁹ Severe infarcts were defined as those with damage to all cell types and complete loss of astrocytic staining within the infarcts by GFAP staining. Moderate infarcts had severely damaged neurons, with moderate damage to astrocytes, with incomplete loss of GFAP staining. Mild infarcts contained ischemic neurons, but astrocytes were normal by hematoxylin and eosin and GFAP staining was either normal or increased.

We outlined the infarct area manually using the auto-outline tool and performed cell counts using a combination of autoscan and manual cell counting modes, with two investigators (K.L. and J.S.T.) confirming each cell count. The outline was drawn at the interface between normal tissue and any ischemic or necrotic neurons. In the severe infarcts the transition from normal to infarcted tissue corresponded with the loss of GFAP staining. Because many infarcts were heterogeneous, with areas of higher and lower cellularity, cell counts were done in both the maximal and minimal areas of cellularity within each infarct. Maximal and minimal cell densities were then calculated, and statistical analysis was performed.

The means and standard deviations presented in the tables were computed for all the infarcts within a severity level or location for both the males and females. To assess the difference in cell density between males and females while adjusting for the influence of infarct size, ANCOVA was used. This procedure assumes that the data are reasonably normally distributed and that the slope between cell density and infarct size is the same for males and females. The assumption of normality was satisfied throughout, but the assumption of equal slopes was not. We therefore used two approaches to analysis.

For those cases in which the assumption was satisfied, a model was fit assuming equal slopes. The probability values presented in Tables 3 and 4 are for intercept and slope. The probability value for slope tests the assumption of equal slopes, and a value of P>.05 supports the ANCOVA approach. The test of the intercept tests whether the adjusted mean cell density is different for males and females when the adjustment is for infarct size. The difference in intercepts estimates the difference in adjusted gender group means.

View this table: [in this window] [in a new window]   Table 3. Comparisons of High Cell Density in Infarct in Male and Female Rats After Embolic Stroke

View this table: [in this window] [in a new window]   Table 4. Comparisons of Low Cell Density in Infarct in Male and Female Rats After Embolic Stroke

When the assumption of equal slopes is not satisfied, as indicated by a value of P<.05, two separate regression lines for males and females are presented. The test of intercept in these cases tests whether the gender group cell densities are different for an infarct size of 0. For these cases we examined the fitted lines to see if the estimated cell densities of one group were larger than the others in the range of interest.

For all analyses we used the generalized estimating equations²³ approach to the regression analysis. This approach models the dependency of the multiple measurements within an animal while estimating across all animals.

	Results

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There were 69 infarcts in female rats, with 54 in males. No significant neurological deficits were observed in the animals other than Horner's syndrome on the right, as previously reported in this experimental model.²⁴ The mortality in female rats was 8%, from anesthetic and the photochemical experiment. This does not differ from our long-term experience with young male rats (N.F., unpublished data, 1990). The severe infarcts were the largest infarcts in both males and females compared with the sizes of the moderate and mild infarcts (Table 1). The mean size of severe infarcts in males was larger than in females. This difference between males and females was most pronounced in the cortex.

View this table: [in this window] [in a new window]   Table 1. Comparisons of Infarct Size in Male and Female Rats After Embolic Stroke

The inflammatory response (Figure), based on cell density, was greater in female rats (Tables 2 through 4). This was true if we evaluated the maximal (Table 3) or minimal (Table 4) cell density. In severe infarcts the maximal cell density was in the periphery of the infarct, with the minimal cell density in the center. In moderate or mild infarcts the pattern of cell density was variable.

View larger version (0K): [in this window] [in a new window]   Figure 1. A, Glial fibrillary acidic protein of a severe deep infarct in a young male rat demonstrates loss of astrocytes within the infarct, with hypertrophy of astrocytes adjacent to the infarcted tissue. Bar=200 µm. B, High-power magnification of a severe cortical infarct in a female rat contains marked inflammatory cells. Most of these cells appear to be macrophages, on the basis of the large amount of cytoplasm and eccentric nuclei (arrows). Bar=50 µm. C, High-power magnification of a similar cortical infarct in a male rat. A few macrophages are present (arrow), with other cells being possibly monocytes or less active macrophages or microglia, along with endothelial cells. Bar=50 µm.

View this table: [in this window] [in a new window]   Table 2. Description of High and Low Cell Density in Infarcts in Male and Female Rats After Embolic Stroke

The most pronounced gender difference in the inflammatory response was in severe infarcts, with both maximal and minimal cell density higher in females. For minimal cell density the groups differed by 1.23x10³/mm², while for maximal cell density the difference was 2.42x10³/mm² initially, with greater differences for larger infarct sizes. For maximal cell density the differences for both moderate (0.99x10³/mm²) and mild (1.04x10³/mm²) infarcts were statistically significant, with females having higher responses. For minimal cell density there was no difference in response between the genders for moderate infarcts. The responses in mild infarcts differed by 1.56x10³/mm² initially and became more similar as infarct size increased. The predicted response for males and females was the same for infarct size of 2.37 mm². There was a tendency for gradual increase in inflammatory response with increasing infarct severity within each gender, with a trend toward higher cell counts in females in moderate and mild infarcts (Table 2) for maximal cell density.

Gender-related differences in inflammatory response for maximal cell density were significant in the cortex. The two groups were significantly different at low infarct sizes (1.19 mm²), and females showed greater differences compared with males as infarct size increased. This trend was also evident in the hippocampus. There was no clear trend in the basal ganglia.

The minimal cell response was similar in the cortex and hippocampus as well. Females had higher responses at low infarct sizes, and these differences were statistically significant; the responses were closer with increasing infarct size. However, the female response was higher over most of the range of infarct sizes for both cortex (equal at 5.29 mm²) and hippocampus (equal at 2.54 mm²). No differences between the genders were noted in the basal ganglia.

	Discussion

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The results confirmed our hypothesis that infarcted tissue in the brain would produce a stronger inflammatory response (cellular infiltrate) in females, consistent with increased immune responses in young females.^{12 14} Although immunohistochemical stains to determine the cell types within the inflammatory infiltrate were not done, the light microscopic appearance is consistent with mononuclear phagocytes. These are most likely activated macrophages but could also be microglia, which derive from the same progenitor cells.²⁵ The differentiation between macrophages and microglia is still subject to some debate.²⁶ Macrophages may participate in tissue damage by releasing toxic substances, including superoxide²⁷ and inflammatory cytokines.^{28 29} This, together with data that macrophage inhibition reduces ischemic damage (as measured at 24 to 48 hours),³⁰ has led to the postulate that macrophages may worsen ischemic damage. Our results, with smaller infarcts in the female rats with the stronger macrophage response, do not support the hypothesis that the macrophage increases tissue damage.

An alternate explanation is that the male animals initially suffered a more severe ischemic insult, leading to larger infarcts. This could be explained by gender-related differences in platelet aggregation. Rosenblum et al³¹ found that testosterone enhanced photochemically induced platelet aggregation when administered to male mice. Interestingly, testosterone did not produce the same effect when given to female mice. In another study estradiol enhanced photochemically induced platelet aggregability in both male and female mice 2 months of age but not in mice 4 to 6 months of age.³² It is possible that the male animals had increased platelet aggregation in response to the photochemical lesion, producing larger strokes.

The gender of experimental animals may influence the results of studies of cerebrovascular disease by multiple mechanisms. Macrophages from male animals produce more hydrogen peroxide,³³ producing more tissue damage than macrophages from female animals. Interleukin-1 (IL-1) genes are expressed early in ischemic tissue.^{34 35} IL-1 stimulates hypothalamic tachykinins in castrated, but not intact, male rats.³⁶ The activity of IL-1, which is a highly inflammatory molecule,³⁷ is inhibited in the brain by high concentrations of estrogen.³⁸ The effect of this gender-related inhibition of IL-1 on stroke is highly complex, since IL-1 is toxic for some cells and growth promoting for others.³⁹

In addition to differences in tissue damage, there are important gender-related differences in responses to potential therapeutic agents. The ability of actinomycin to induce the production of nerve growth factor in the submaxillary gland of mice is decreased in male animals in the presence of testosterone.⁴⁰ The biological half-life of corticosterone is shorter in female than in male rats,⁴¹ suggesting a need for alternate dosing regimens to produce equivalent therapeutic effects in males and females. Female rats have a more pronounced immunosuppressive effect from antithymocyte globulin than males.¹⁴ It is clear that these complex interactions between gender and tissue damage after stroke will be elucidated only if both male and female rats are used in stroke research.

Experimental stroke research has been performed almost exclusively on young male animals. The reasons for the use of male animals to the exclusion of female animals are not clear. One possibility is the misconception that female animals are more expensive than male animals. If male and female animals of the same weight are purchased, it is true that the female animals are more expensive. If animals are ordered by age, the female animals will weigh less than the male animals, but the cost is identical. When we compare the design of studies in animals with those in humans, it is interesting to note that the "Methods" section of animal studies lists the weight rather than the age of the animals. A similar practice in humans could lead to potential studies of "50-lb children." Such a study design would be considered scientifically unacceptable in humans. It is surprising and dismaying that basic principles of study design (such as age and gender of the experimental animal) have not received adequate consideration in animal research.

A legitimate problem in using female animals is variability of hormonal levels at different points in the estrus cycle.^{42 43} Although this complicates research, care of women is similarly complicated by the estrus cycle. This problem could be minimized in stroke research by using postmenopausal female animals, which would correspond better with the age and hormonal status of most women at risk for stroke.

We conclude that gender may influence the inflammatory response after stroke. Our data and other evidence from the literature that gender may alter tissue damage and therapeutic responses suggest that adequate understanding of stroke in women will require the use of female animals in stroke research.

	Acknowledgments

 
This study was sponsored by Public Health Service grant RO1AG11759. The authors wish to thank Sheryl Orok and Lori Wolenski for assistance in preparation of the manuscript.

Received March 20, 1995; revision received December 7, 1995; accepted December 13, 1995.

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