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(Stroke. 1996;27:1663-1668.)
© 1996 American Heart Association, Inc.

Articles

Effect of Age in Rodent Models of Focal and Forebrain Ischemia

Garnette R. Sutherland, MD; Gary A. Dix, MD Roland N. Auer, MD, PhD

the Departments of Clinical Neurosciences (G.R.S., G.A.D., R.N.A.) and Pathology (R.N.A.), University of Calgary (Canada).

	Abstract

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Background and Purpose The majority of animal experiments examining the nature and treatment of stroke have used relatively young animals ranging in age from 2 to 6 months. However, significant morphological, neurochemical, and behavioral changes occur with aging in rodents, particularly during the first 24 months of age. This study examines the effect of age in two models of transient ischemia, a forebrain and a focal model, in male Wistar rats.

Methods We induced forebrain ischemia of 12 minutes' duration by bilateral carotid artery occlusion with controlled hypotension at a mean blood pressure of 45 mmHg and, using an intraluminal filament technique, induced focal middle cerebral artery occlusion of 100 minutes' duration at a mean blood pressure of 60 mm Hg. Physiological parameters were monitored and maintained within normal limits. On day 7 after ischemia, the rats were perfusion-fixed and the brains removed for quantitative histopathology.

Results After forebrain ischemia, older rats showed significantly less CA1 neuronal necrosis than the younger group (P<.003), whereas both striatal and neocortical injury were significantly greater in the older group (P<.05). Among animals subjected to focal ischemia, the volume of infarcted tissue and the number of necrotic neurons in the area adjacent to the infarction were both greater in older rats (P<.05).

Conclusions This study emphasizes the importance of age in models of forebrain and focal ischemia. The interaction between age-related changes in morphology, neurochemistry, and behavior on the ischemic cascade complicates the interpretation of mechanistic data, and pharmacological effects observed in younger animals may not necessarily translate to an older population.

Key Words: aging • animal models • cerebral ischemia, focal

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References Introduction

 
Although human stroke occurs at all ages, a limited number of reports have examined the effect of age in models of cerebral ischemia. Focal ischemia studies have shown infarct volume to be greater in older animals.^{1 2} Forebrain ischemia experiments have been restricted to spontaneously hypertensive rats, in which an older group showed greater injury than a middle-aged group.^{3 4} Further studies examining the effect of age in rodent models of cerebral ischemia are relevant because the effect of age may be nonlinear, dependent on region, or variable between rodent strains.

Laboratory animals undergo aging within a compressed time scale, and although age-related microcirculatory changes have been defined in laboratory rodents, they do not develop atherosclerosis.^{3 5} A primary age-related susceptibility of the brain to ischemic injury can thus be more easily examined with animal models. This study evaluates the effect of age on ischemic brain injury in adult male Wistar rats in both focal and forebrain ischemia models.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References Introduction

 
Animal Preparation and Experimental Groups
All animals were cared for in accordance with the guidelines of the Canadian Council on Animal Care. Fifty-two male Wistar rats were used for this study; 27 were classified as young (2 to 3 months), and 25 were old (26 to 28 months). The animals were fasted for 24 hours before being used in the experiments, during which they were allowed free access to water. The rats were randomly divided into two groups, with 30 rats (15 young and 15 old) to be subjected to transient forebrain ischemia and 22 rats (12 young and 10 old) to undergo MCA occlusion. Each rat was intubated under 3% to 4% halothane anesthesia and kept mechanically ventilated for the duration of the surgery to maintain normal arterial blood gases. Brain temperature was monitored before, during, and after the ischemic insult with a tympanic membrane probe and was maintained at 37.5±0.1°C with a thermoregulated, servo-controlled, heated water blanket and/or an overhead heating lamp.

The tail artery was exposed and cannulated to obtain samples for blood gases, hematocrit, and blood glucose measurements and for blood pressure monitoring and control throughout the experiment. Maintenance fluids (0.9% normal saline) were administered via a jugular vein or tail artery at a constant rate of 4 mL/kg per hour during the operative procedure. Body weight was determined before ischemia and 1 week later at the time of perfusion fixation.

Induction of Transient Forebrain Ischemia
A modification of the model developed by Siesjo and coworkers⁶ was used. After exposure of the carotid arteries through a neck incision, transient forebrain ischemia was induced by bilateral carotid artery occlusion coincident with a reduction in systemic blood pressure to a mean of 45 mm Hg through aspiration of blood into a heparinized syringe. At this blood pressure and temperature (37.5°C), 12 minutes of occlusion has been found to result in consistent damage. After 12 minutes, blood flow was restored and the shed blood was reinfused. After wound closure and the discontinuation of halothane anesthesia, the animals were allowed to awaken.

Induction of Reversible Focal Ischemia
MCA occlusion was accomplished by the intraluminal thread technique.⁷ The ECA was first isolated and divided through a ventrolateral neck incision; a temporary clip was placed at its origin, and a 6-0 silk suture was tied loosely around the ECA stump. The stump was then cut, and a 22-mm 3-0 monofilament nylon suture was introduced into the ECA; at the same time, the silk suture was tightened to prevent bleeding, which permitted removal of the temporary clip. The nylon suture was advanced into the internal carotid artery via the ECA until faint resistance was encountered and a sharp increase in monitored blood pressure was noted, which indicated that the suture tip was situated in the proximal segment of the MCA. Before MCA occlusion, mean systemic blood pressure was reduced to 60 mm Hg by adjusting the depth of halothane anesthesia and was maintained at that level until the nylon suture was removed after a total occlusion time of 100 minutes; we have previously demonstrated that this combination of parameters results in an infarct that is consistent and relatively large.⁸ After the discontinuation of halothane and wound closure, the animals were allowed to awaken.

Histopathology: Forebrain Ischemia Model
Rats in the forebrain ischemia group were allowed to survive for 7 days after the ischemic insult. Rats that died before this time were excluded from the histopathologic analysis but were used in mortality calculations.

Each rat was perfusion-fixed with 1 L of 4% phosphate-buffered formaldehyde at pH 7.3. The brains were removed 12 hours later and were cut coronally into 3-mm-thick slices and embedded in paraffin. Sections 8 µm thick were cut and stained with hematoxylin and eosin or immunohistochemically prepared for GFAP (GFAP antiserum, 1:500 dilution, Dako, Dimension Laboratories). Quantification of ischemic neuronal injury was performed on standardized sections of cerebral cortex, hippocampus, and striatum. In the neocortex and striatum, the absolute number of ischemic neurons was counted in both the left and right hemispheres. Quantification of ischemic neurons in the pyramidal layer of the CA1 zone of the hippocampus was performed by one individual at two hippocampal levels (bregma -3.4 and -5.0) of both hemispheres. We calculated the frequency of ischemic neurons by dividing the number of acidophilic and/or pyknotic neurons by the total number of neurons to obtain the percent necrosis. The number of GFAP-positive astrocytes in the hippocampus was determined by direct counting of the anterior hippocampal section, where damage was severe.

Histopathology: Focal Ischemia Model
Rats in the focal ischemia group were allowed to survive for 7 days after the induction of ischemia. Rats that died before this time were excluded from the histopathologic data analysis but were used in mortality calculations.

Each rat was perfusion-fixed with 4% phosphate-buffered formaldehyde at pH 7.3. The brain was removed 12 hours later and cut coronally into 3-mm slices. After they were processed and embedded in paraffin, the brain slices were sectioned serially at 8-µm thicknesses, stained with hematoxylin and eosin, and prepared on slides at 250-µm intervals. Rostral and caudal limits were bregma +5.2 mm and -6.8 mm, respectively. A total of 16 coronal sections were examined for each rat. All sections were evaluated qualitatively by one individual, and the topographic extent of brain damage was quantitated. A video analysis system (JAVA, Jandel Scientific) was used to demarcate infarct borders and trace a series of four polygons: one comprising necrotic neocortical tissue, one of striatal necrosis, and two additional polygons comprising ischemic and contralateral hemispheres. These tracings were included to more accurately characterize the volume of tissue loss, given the observation of obvious atrophy in the ischemic cerebral hemisphere compared with the undamaged hemisphere. We used a three-dimensional reconstruction computer program to determine the volume of hemispheric tissue loss, using the totals calculated through the summation of cortical necrosis, striatal necrosis, and atrophy. We controlled for variable brain size between samples by expressing infarct size as a percentage of the contralateral hemisphere.

Examination of the neocortical sections revealed a histological penumbra of selective neuronal necrosis. The penumbra was quantitated by direct visual counting of the absolute number of acidophilic and/or pyknotic neurons, irrespective of the distance from the infarct, in the coronal section at bregma -1.3 mm.

Statistical Analysis
All results are presented as mean±SEM. Statistical analysis for both experiments was conducted with a one-way ANOVA test, with arcsine transformation of data expressed in percentages. Statistical significance was taken at the P<.05 level.

	Results

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Forebrain Ischemia Model
Physiological data for groups subjected to transient forebrain ischemia are presented in Table 1. Other than body weight, PaO₂, and hematocrit, there were no significant differences between young and aged rats for any of the parameters. Preischemic and postischemic PaO₂ and hematocrit values were significantly less in young versus old animals (P<.01). Body weight was greater in older rats than in young rats (P<.01).

View this table: [in this window] [in a new window]   Table 1. Physiological Parameters in Forebrain Ischemia

Of the 30 rats subjected to forebrain ischemia, only one young rat (3% of the total group) died before perfusion fixation. Older rats sustained less damage in the CA1 hippocampus than young rats (Figs 1 and 2). In the anterior hippocampal region (bregma -3.4), the percentage of necrotic cells was less in old than in young animals (43.4±6.6% and 63.8±4.5%, respectively; P<.003). This was also observed in the posterior hippocampus (bregma -5.0), with a statistically significant reduction (P<.0009) in the degree of ischemic damage in the older rats (41.1±6.4%) compared with the young animals (61.6±4.3%). In the striatum (Fig 2), the older animals showed a greater degree of neuronal damage than their younger counterparts (35.9±13.5 versus 11.23±4.1; P<.05). Similar to the striatum, in the neocortex (Fig 2), older rats demonstrated a greater number of necrotic neurons than younger rats (131.0±40.3 versus 29.4±12.3; P<.02). The degree of reactive gliosis in the anterior hippocampus, as determined by the number of microscopically visualized astrocytes on GFAP-stained slides, did not differ significantly between the two groups (150±17 per anterior hippocampal region in old rats compared with 154±12 per anterior hippocampal region in young rats).

View larger version (264K): [in this window] [in a new window]   Figure 1. Photomicrographs of CA1 sector, 1 week after forebrain ischemia, in a young rat (top) and an old rat (bottom) show less injury in the pyramidal cell band of the older animal (original magnification x200).

View larger version (39K): [in this window] [in a new window]   Figure 2. Quantitative histopathology, forebrain ischemia. Shown are number of necrotic neurons per section, extending from the cingulate gyrus to the entorhinal sulcus (neocortex), total number of necrotic neurons in a standard striatal section, and the percent hippocampal necrosis at two levels (right). Ant indicates anterior. *P<.05, **P<.005, ***P<.001.

Focal Ischemia Model
Physiological parameters for groups subjected to transient focal cerebral ischemia are presented in Table 2. Other than body weight and PaO₂, there were no significant differences between young and aged rats for any parameters. Preischemic and postischemic PaO₂ values were significantly greater in young rats than in old rats (P<.01). Body weight was greater in older rats than in younger rats (P<.01).

View this table: [in this window] [in a new window]   Table 2. Physiological Parameters in Focal Ischemia

Among animals subjected to focal ischemia, 3 (14%) died (2 young, 1 old) before perfusion fixation. The animals demonstrated predominantly striatal and neocortical damage, with the volume of necrotic striatum in old rats amounting to 58.6±4.5 mm³ compared with 38.1±5.6 mm³ in young animals. When expressed as a percentage of the total hemispheric volume (Fig 3), striatal necrosis constituted 11.5±0.8% and 8.0±1.2%, respectively (P<.03). Neocortical infarction was marginally smaller, expressed as a percentage of the hemisphere in young animals, but this was not statistically significant (Fig 3). An increase in the number of necrotic neurons in the penumbra of the infarcted tissue was noted at bregma -1.3 mm in older rats (2540±171 versus 1771±308 neurons; P<.05).

View larger version (45K): [in this window] [in a new window]   Figure 3. Quantitative histopathology, focal ischemia. Brain damage is expressed as percentage of the opposite hemisphere. Both pannecrosis (infarction) in the cerebral cortex and striatum and selective neuronal necrosis are augmented with age. *P<.05.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References Introduction

 
Global Cerebral Ischemia and Age
The novel and unexpected observation in this experiment was that forebrain ischemia was associated with an age-dependent, regional vulnerability. In the CA1 sector of the hippocampus but not in other brain regions, necrosis was less in aged rats. This contrasts with a previous report³ in which aged spontaneously hypertensive female rats showed significantly greater neuronal damage to CA1 pyramidal neurons of the hippocampus after 20 minutes of forebrain ischemia than a younger group, despite no greater decline in CBF in older rats.

Focal Cerebral Ischemia and Age
In this study a significant difference between infarct size and distribution between young and old male Wistar rats was observed. Despite the fact that we controlled for hemispheric size by expressing infarct size as a percentage of the hemisphere, age was associated with an increase in infarct size from 9% to almost 12% in the neocortex and from 8% to almost 12% in the striatum. One previous study in male Wistar rats, in which permanent rather than transient focal ischemia was used, demonstrated 40.5% hemispheric infarction in aged (28 to 36 months) rodents and 30.9% infarction of the hemisphere in young (11 to 17 months) animals,¹ a percent increase with aging comparable to that seen in the present study. Another study of the effect of aging in several rat strains found no exacerbation of infarct size with age.⁹ A third study was unable to produce MCA occlusion in aged rats with the intraluminal suture technique.¹⁰ A fourth study did not measure infarct size directly but found decreased macrophage infiltration in older rats subjected to embolic occlusions.²

Aging, CBF, and Ischemia
Changes in the microcirculation of the aging rat brain include decreased capillary lumen diameter and decreased capillary endothelial cells, and increased microvascular tortuosity¹¹ and deformity^{5 12} and vascular thickening due to increased intramural collagen, flocculent material, and pigment deposition.¹³ These structural changes in the vasculature may explain the impairment of cerebral autoregulation seen with aging.¹⁴

CBF may show a slight decline with aging,¹⁵ and MCA occlusion leads to a greater ischemic fall in CBF in aged animals.¹ With the use of hypotension in the present study, age-related impairment in autoregulation might be expected to augment differences in CBF and infarct size between the young and old groups.

In the two-vessel occlusion forebrain ischemia model, aging leads to no greater fall in hippocampal blood flow below resting values.^{3 16} While the somewhat higher hematocrit levels in the older group could be associated with compromised CBF, this would not account for the observed regional dependence of ischemic neuronal injury with less hippocampal injury in the older group. As in the pathophysiology of cerebral ischemia itself, parenchymal as well as vascular factors need to be considered in attempting to explain age-related changes in vulnerability.

Brain Parenchymal Changes With Aging
The majority of investigations have shown an age-related decline in cortical and hippocampal neuronal density and number in the rodent^{17 18} and human^{19 20} brain, although new counting methods have cast doubt on the dogma of age-related neuronal loss.²¹ Most brain regions show reduced dendritic fields.^{22 23} Excitotoxicity likely constitutes a component of ischemic brain damage, but age-related changes in excitotoxic damage are not consistently reported. Although one report suggested that aged rats have diminished excitotoxic damage after intrahippocampal injections of kainic acid,²⁴ other animal studies²⁵ and human experience²⁶ suggest that susceptibility to excitotoxicity is enhanced with age.

Age-related decreases have been reported in the number of kainate, NMDA, and AMPA membrane receptors in cortex and hippocampus, with a selective age-related impairment in the NMDA system.^{27 28 29} Although numbers of NMDA receptors may decrease with age, the functional sensitivity of NMDA receptors could be enhanced with age.³⁰ It is thus difficult to explain age-related changes in ischemic vulnerability on the basis of atypical excitatory neurotransmitter receptor systems.

Aging, Cerebral Metabolism, and Free Radicals
The local cerebral metabolic rate for glucose decreases between 3 and 12 months of age in rats.^{31 32} Mice³³ and humans³⁴ show a similar age-related decline.

Brain protein synthesis is reduced with aging,³⁵ but damage to existing proteins also increases. The activity of numerous enzymes declines with age, including hexokinase, phosphofructokinase, and phosphoglyceromutase,³⁶ and inactive forms of 3-phosphoglycerate kinase are known to accumulate in the brains of aged rats.³⁷ All these changes may be related to free radical–induced protein oxidation with aging,^{38 39} yielding carbonyl groups,^{40 41} a process that also occurs in ischemia itself.^{42 43} If free radical generation is responsible for initiating such neuronal damage,⁴⁴ it could be exacerbated by specific oxidative metabolic dysfunctions associated with aging.⁴⁵ Enzymatic free radical scavenging systems are less effective with advancing age, resulting in the cytosolic accumulation of oxygen-derived free radicals, leading to degradation of mitochondrial DNA and cellular membranes, as a result of lipid-peroxidative destruction.^{5 46} If free radicals detrimentally influence the outcome of stroke, as has been suggested,^{44 47 48} then age-related deficiency in the handling of toxic free radicals may provide one possible explanation for a poorer outcome of cerebral ischemia in the aged.⁴⁹

Summary
In summary, although the mechanism(s) of age-related ischemic susceptibility has not been defined by the present study, the results emphasize the importance of age in models of cerebral ischemia, particularly because the effect of age may be nonlinear and region-specific.

	Selected Abbreviations and Acronyms

 

AMPA = -amino-3-hydroxy-5-methyl-4-isoxazolepropionate CBF = cerebral blood flow ECA = external carotid artery GFAP = glial fibrillary acidic protein MCA = middle cerebral artery NMDA = N-methyl-D-aspartate

	Acknowledgments

 
This study was supported by grants from the Heart and Stroke Foundation of Canada (Drs Sutherland and Auer). The authors acknowledge the technical support provided by Dr Fang-Wei Yang and Yu Zhi Wang. Appreciation is also extended to Maureen Firmston and Florence Yang for preparation of the manuscript.

	Footnotes

 
Reprint requests to Dr Garnette R. Sutherland, Department of Clinical Neurosciences, Division of Neurosurgery, Foothills Hospital, 1403-29 St NW, Calgary, Alberta T2N 2T9, Canada. E-mail garnette@indy.dcns.ucalgary.ca.

Received November 10, 1995; revision received May 13, 1996; accepted June 5, 1996.

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Editorial Comment

Raymond C. Koehler, PhD, Guest Editor

Department of Anesthesiologyand Critical Care MedicineThe Johns Hopkins UniversityBaltimore, Md

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References Introduction

 
Clinical stroke is more common in the elderly, yet most experimental studies of stroke use young animals. In the present study the authors evaluated whether neuronal injury is greater in older rats (26 to 28 months) than in younger rats (2 to 3 months). The strengths of the study include the use of both focal and global models of ischemia and detailed counting of necrotic neurons in various regions.

With 100 minutes of MCA occlusion, aged rats had a larger infarction in striatum and a greater number of necrotic neurons in the cortical penumbra. However, to reduce the variability in infarct size, the inspired halothane concentration was increased to reduce arterial pressure to 60 mm Hg during the 100-minute period of ischemia. It is possible that the reduction in intraischemic blood flow was greater in aged rats if halothane selectively impaired autoregulation in aged rats or if autoregulatory capacity is inherently less in aged rats. Therefore, although these results are consistent with the hypothesis that neurons in aged rats are more vulnerable to ischemia, the possibility that the ischemic insult was not matched between age groups cannot be excluded.

With forebrain ischemia involving carotid artery occlusion plus hypotension, blood flow is known to decrease to near-zero levels, and the ischemic insult is presumably well matched between age groups. Because arterial pressure is similar during early reperfusion, capillary reflow is assumed to be similar, although this was not investigated. Thus, the greater number of necrotic neurons in cortex and striatum observed 1 week after forebrain ischemia in aged rats most likely represents increased vulnerability in these neuronal populations.

The most remarkable finding of the study was that the percentage of pyramidal neurons that were necrotic in the CA1 region of hippocampus was actually less in the aged rat. One possibility is that these neurons receive less excitatory input during reperfusion in the aged rat because of increased vulnerability of other neurons involved in CA1 connectivity. Another consideration is that although histology was performed at 7 days, maturation of necrosis conceivably could extend beyond 7 days in the aged rat. Alternatively, the aging process itself may have caused selective loss of the pyramidal neurons that would be most vulnerable to ischemia, although the issue of whether there is loss of these neurons with aging is still debatable.

The mechanisms of age-dependent changes in ischemic vulnerability remain speculative at this time. Age-dependent decrease in antioxidant defenses is one potential mechanism. The present results indicate that one must consider the nature of the ischemic insult, regional differences, and possibly neuronal connectivity in interpreting the results. Finally, as with all studies of aging based on rodents with life spans of a few years, caution is required before results are extrapolated to aging processes in humans.

	Selected Abbreviations and Acronyms

 

AMPA = -amino-3-hydroxy-5-methyl-4-isoxazolepropionate CBF = cerebral blood flow ECA = external carotid artery GFAP = glial fibrillary acidic protein MCA = middle cerebral artery NMDA = N-methyl-D-aspartate

*P<.01 compared with young rats.

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