Interaction Between a Rat Model of Cerebral Ischemia and β-Amyloid Toxicity
Background and Purpose— Clinical data suggest that Alzheimer disease (AD) and stroke together potentiate cognitive impairment. Inflammatory mechanisms are involved in AD pathology and stroke and may be the mediator between AD and stroke toxicity.
Methods— AD was modeled by cerebroventricular injections of β-amyloid (Aβ[25–35]) and subcortical lacunar infarcts by striatal endothelin injections. Inflammatory mechanisms were examined using immunohistochemical analysis. Memory and motor tasks were assessed using the Montoya staircase test.
Results— Aβ injections elicited increases in pathological and inflammatory correlates of AD in multiple forebrain sites. Increases in astrocytosis and reactive microglia in the hippocampus were enhanced with the combination of endothelin and Aβ(25–35). Aβ(25–35) treatment decreased performance in the Montoya staircase behavioral test.
Conclusions— The enhanced inflammatory response with Aβ toxicity and ischemia may mediate the inability to improve behavioral performance caused by the stroke. Anti-inflammatory treatment may ameliorate the pathological and behavioral deficits associated with the combination of AD and stroke.
Neuropsychometric tests of cognitive function have shown that patients with autopsy evidence of Alzheimer disease (AD) pathology and cerebral infarcts, especially lacunar infarcts in the striatum, show more cognitive impairment than patients with AD pathology alone.1,2 Ischemic brain insults increase neurofibrillary tangle formation3 and amyloid precursor protein (APP),4,5 suggesting a possible link between cerebral ischemia and AD.
Inflammatory events in the brain are closely associated with AD and cerebral ischemia, including the upregulation of cytokines, their respective receptors, and the transcription factor regulating many inflammatory molecules, nuclear factor κB (NFκB).6 Reactive astrogliosis and microgliosis are also often found in areas of β-amyloid (Aβ) plaque deposits and ischemic regions.7 Thus, inflammatory events that occur in the AD brain may be involved in the neurotoxicity caused by plaques, tangles, and/or the spreading of AD that gives the disease its characteristic neurodegenerative property.
The combination of AD and cerebral ischemia may increase overall inflammatory damage in the brain, leading to increased neurodegeneration. To examine this possibility, AD pathology was generated using intracerebroventricular injections of Aβ(25–35), a toxic fragment of the Aβ, alone or in combination with striatal injections of the potent vasoconstrictor endothelin9 to mimic the subcortical lacunar infarcts seen clinically. Pathological correlates of AD and inflammation were assessed using histochemical and immunohistochemical analysis. The Montoya staircase test8 was used to examine motor and/or learning deficits.
Materials and Methods
Surgery (Day 0)
All experimental procedures were performed according to the animal care guidelines of the University of Western Ontario. Male Wistar rats (250 to 300 g; Charles River, Montreal, Quebec) were divided into 4 groups: intracerebroventricular injections of Aβ(25–35) (BACHEM, Torrance, Calif); endothelin injections into the right striatum; both Aβ and striatal endothelin injections; and sham procedures. The Aβ(25–35) fragment was used to reduce the possibility of rapid coagulation and to allow diffusion of the peptide into the brain.
All rats were anesthetized using 40 mg/kg of pentobarbital (Somnotol) intraperitoneally. Body temperature was maintained at 37°C. Aβ(25–35) (50 nmol in 10 μL of saline) was injected bilaterally into the lateral ventricles. Details on Aβ(25–35) composition and preparation are described elsewhere.9 Aβ(25–35) (50 nmol in 10 μL of saline at a rate of 1 μL/min) was injected bilaterally into the lateral ventricles via a stainless-steel cannula (23-gauge; anterior/posterior −0.8, medial/lateral ±1.4, and dorsal/ventral −4.0 below dura). Two endothelin injections (6 pmol in 3 μL of saline), 1 mm apart, were made into the right striatum.9 Sham procedures involved all the surgical steps without injections of Aβ(25–35) or endothelin. After wound suture, all rats received 40 mg/kg of buprenorphine intramuscularly and were allowed to recover from surgery for 7 days.
For training (n=10 for each group), rats were placed into the Montoya staircase apparatus8 at the same time every day for 8 days (days −8 through −1) and were allowed to perform the test for 20 minutes. Rats were food-deprived for 1 day (day −9) before testing, as well as on day −8. On days −7 though −1, rats were fed 10 grams of their normal chow so that they maintained their body weight at ≈85%. Three Noyes precision food pellets (Research Diets Inc) were placed in each of the 14 wells (7 on each side). Pellets eaten were recorded from each well. Re-testing trials, which were performed 7 days after the surgical procedures (day 0), were performed for 8 days in the same way as the training trials (days 8 through 15).
Twenty-one days after surgery, all animals were euthanized via pentobarbital overdose and perfused transaortically first with saline followed by 4% formaldehyde (pH 7.4). The brains were removed and cryoprotected in 30% sucrose for 36 hours at 4°C. Coronal sections (30 μm) were cut using a cryostat.
Thionine and Congo Red staining procedures were performed after sections being mounted on slides.10
Free-floating sections from rat brains (n=5 for each group) were treated with 0.03% hydrogen peroxide and blocked with 3 mL of horse or rabbit serum (Vectastain Elite, Vector Laboratories, Burlingame, Calif). The following primary antibodies diluted in filtered phosphate buffered saline: APPa4 mouse monoclonal (Chemicon International Inc, Temecula, Calif; 1:1000), GFAP mouse monoclonal (Sigma-Aldrich, St Louis, Mo; 1:1000), OX-6 mouse monoclonal (Serotec Inc, Raleigh, NC; 1:1000), Tau-2 mouse monoclonal (Sigma-Aldrich, St Louis, Mo; 1:10000), NFκB(p65) goat polyclonal (Santa Cruz Biotechnologies, Santa Cruz, Calif; 1:1000), IL-1β goat polyclonal (Santa Cruz Biotechnologies, Santa Cruz, Calif; 1:1000), and TNF-α–M18 goat polyclonal (Santa Cruz Biotechnologies, Santa Cruz, Calif; 1:1000). APP-, Tau-2–, GFAP-, and OX-6–stained sections were incubated with 3 mL of horse biotinylated anti-mouse secondary antibody and horse serum. NFκB(p65), IL-1β, and TNF-α–stained sections were incubated with 3 mL of biotinylated rabbit anti-goat secondary antibody and rabbit serum. Sections were then incubated in 3 mL of avidin–biotin complex followed by 0.05% diaminobenzidine. Sections were washed, air-dried, cleared in xylene, and cover-slipped. Brains to be compared were processed at the same time using the same solutions to reduce variability in immunostaining caused by separate processing.
Digital photographs were taken using a light microscope (Leitz Diaplan). An investigator was blinded to the identification of the rat sections while analysis was being performed. All immunohistochemical data that were compared was processed at the same time to reduce any variation caused by differences of intensity. A grading scale was based on both observable size and intensity of staining (n=5 for each group). Also, relative optical density measurements and numbers of stained cells or accumulations were measured in entire brain areas for quantitative analysis and statistically analyzed with ANOVA and Tukey post hoc test with a significance level of P≤0.05. Statistical analysis, using the Student t test, was performed on the staircase test data with a significance level of P≤0.05. The staircase data are expressed as the mean±SEM.
Endothelin-induced ischemia resulted in an extensive increase in immunostaining in the right striatum in the region of the infarct (Table I and II, available online only at http://www.strokeaha.org). Reactive astrocytes (GFAP) and microglia (OX-6) covered an extensive region surrounding the infarct (Figure 1E and 1G). OX-6–positive stained microglia also covered a large area of the striatum (Figure 1E). Increases in both APP (Figure 1A) and Tau-2 (Figure 1C) immunostaining in the region of ischemic damage were also present. Tau-2 and APP staining showed a similar distribution, intensity, shape, and size, and appear to be both intracellular and extracellular. Increases in NFκB(p65) (Figure 1I), IL-1β (Table I), and TNF-α (Table I) immunostaining showed a punctate appearance in the region of the infarct.
Aβ(25–35) injections increased APP-positive staining in hippocampal and neocortical areas. In the cerebral cortex, single cells and diffuse intense clumps ≈20 to 25 μm in diameter were stained for APP (Figure 2A). APP signal also increased in the corpus callosum (Figure 2C), fornix/anterior hippocampus, and dentate gyrus (Table I and II).
The increased Tau-2 signal in the cortex is both cellular and clustered in appearance (Figure 2E). Tau-2 signal was also present in the corpus callosum (Figure 3G). The CA2 region of the Aβ-treated rat hippocampus showed positive fibrillar type immunostaining for Tau-2 (Table I).
Aβ treatment resulted in reactive microglia and astrocytes in the corpus callosum and cingulate gyrus (Figure 3A and 3C). Activated microglias were also present in the thalamus and cortex (Table I). TNF-α and NFκB signals were also increased in the cortex and corpus callosum of Aβ-treated rats (Figure 3E and 3G)
Combined Aβ(25–35) and Endothelin Injections
In combined Aβ/endothelin-treated rats, there was a greater intensity of staining of both OX-6–positive microglia (Figure 4G) and GFAP-positive astrocytes (Figure 4H) in the hippocampus, particularly in the CA1 region, compared with endothelin or Aβ-treated rats (Figure 4E and 4F). Average intensity measurements for GFAP in the hippocampus of combined Aβ–endothelin rats (191.9±4.1) was significantly higher than sham (148.2±2.2), endothelin (152.9±11.0), and Aβ (162.9±8.7). Similarly, average numbers of OX-6–stained microglia in the CA1 of the hippocampus of combined Aβ–endothelin rats (36.0±6.5) was significantly higher than sham (1.8±0.7), endothelin (4.8±0.9), and Aβ (13.4±5.1). Other proteins showed increased signal in combined Aβ/endothelin-treated rats (Table I and II).
Congophilic Aβ Deposition
Aβ-treated rat brains showed increased proliferation of microglia surrounding the lateral ventricles and in the corpus callosum (Figure 5A) and deposits of congophilic Aβ in the wall of the lateral ventricle (Figure 5B). Rats receiving Aβ also had lesions in the fornix, anterior hippocampus (Figure 5C), and CA1 hippocampus (Figure 5E), which stained amyloid positively with Congo Red (Figure 5D and 5F). The infarct in endothelin-injected rats was surrounded by microglia cells (Figure 5G) and stained positively for Congo Red.
The Staircase Test
After Aβ or the combined Aβ/endothelin injections, there was a significant deficit in the total number of pellets eaten on day 8 compared with the last day of training (day −8), which disappeared by day 9 (Figure 6A). Sham-treated rats did not show a significant deficit on the first day of re-testing in either paw, whereas Aβ-treated rats showed a significant deficit on day 8 for the ipsilateral paw (73±8% of pretreatment). Endothelin-treated rats showed a significant deficit on the first day of re-testing (day 8) for the paw contralateral to the infarct (66±15% of pretreatment; Figure 6B). Although the ipsilateral paw of endothelin treated rats improved performance during the post-treatment phase (143±18% of pretreatment), the combined Aβ/endothelin-treated rats on day 8 showed deficits in both the ipsilateral (79±12% of pretreatment) and contralateral (71±13% of pretreatment) paws and no improved performance over time in the paw ipsilateral to the stroke (Figure 6B). Aβ-treated rats showed a significant deficit on day 8 for the ipsilateral paw (73±8% of pretreatment).
In the critical study linking AD and cerebral ischemia in humans, the strongest correlation was found with lacunar infarcts in the striatum.2 Our focal ischemia model using endothelin was chosen to mimic the multiple striatal infarcts seen in these AD patients.2 Previous studies in our laboratory have shown that reperfusion occurs ≈1 hour after endothelin-induced ischemia and infarct size is reduced by anesthetic administration. Thus, the infarct may be smaller when endothelin is administered in the anesthetized animal as opposed to the awake animal.11 However, the aim of the study was to demonstrate small increases in inflammatory responses in rat models of AD and cerebral ischemia alone and to demonstrate whether there is a synergistic increase in the inflammatory response in a combined rat model of AD and cerebral ischemia. Likewise, to minimize inflammatory damage caused by surgical procedures, sham operations were chosen as a control for our model of AD. In ongoing studies, there were no pathological or behavioral differences between rats treated with the inverse Aβ peptide (Aβ[35–25]) and sham-treated rats. In our rat model of cerebral ischemia, we were able to show increases in the inflammatory response in the striatum. In this study, we have shown TNF-α and IL-1β immunostaining in the area of ischemic damage similar to what has previously been demonstrated.12 The level of signal in our study was relatively low but consistent with previous studies indicating that acute inflammatory responses occur 3 days after the initial insult.13
Of particular interest are the observed increases in both APP and Tau-2 in the region of the infarct. APP levels previously have been found in the region of the infarct after ischemic insult.14 However, chemical alterations in both tau and APP proteins need to occur for neurofibrillary tangles and senile plaques to form. The presence of both these molecules in the region of the infarct suggests a possible link between AD pathology and cerebral ischemia. In addition, APP and tau proteins may have more general roles in mediating neurotoxic and neuroinflammatory events.
Previously, injections of Aβ(25–35) into the rat amygdala induced inflammation and Tau-2 immunostaining adjacent to the amygdala and in some regions with direct connections to the amygdala.9 We have used bilateral injections of Aβ into the lateral ventricles to expose more forebrain regions to the toxic effects of this peptide. Inflammatory responses were observed in diverse sites including cortical, hippocampal, striate, thalamic, and ventral hypothalamic (arcuate nucleus) regions. Increases in total NFκB were observed. To test activated NFκB, a more robust test such as an electrophoretic mobility shift assay is required. Widespread neuropathological changes were observed, including accumulations of microglia cells, often near congophilic Aβ deposits in the corpus callosum, cortex, and hippocampus. Although there is no clinical evidence of Aβ(25–35) fragments in AD brains, Aβ(25–35) administration yielded results similar to many neuropathological aspects of both transgenic models AD and human AD.15–18
This is the first study to our knowledge examining the combined pathological and inflammatory effects of Aβ peptide-induced toxicity and ischemia in the rat. Previous investigations have shown that both acute and chronic injections of Aβ(25–35)19,20 and Aβ(1–40) or Aβ(1–42)21–23 were neurotoxic; however, some questions remain unanswered. The distribution of exogenous Aβ in specific brain sites is not known. Additional experiments are required to determine the extent of the inflammatory response of the rat brain to various concentrations of Aβ injections and/or multiple injections over several days.
The combination of Aβ toxicity with focal ischemia elicited a very strong inflammatory response. Specifically, the hippocampus of the combined Aβ/endothelin-treated rats showed increases in reactive astrogliosis and microgliosis compared with endothelin or Aβ alone. Although lacunar infarcts alone cause dementia, these data may provide some indication of the possible pathological substrates for the finding that increased dementia can be observed in patients with low-level AD and multiple lacunar striatal infarcts.2
Administration of Aβ, with or without endothelin, resulted in a reduction of pellets eaten on the first day of retesting. Although the Montoya staircase test was designed to detect motor deficits,8 these results suggest that the Aβ-treated animals had an impairment in their memory for the task and had to undergo a short relearning phase. This memory deficit is consistent with the pathological and inflammatory changes observed in the hippocampus.
Endothelin-treated rats showed a significant reduction in pellets eaten by the paw contralateral to the infarct only, and the paw ipsilateral to the infarct showed an improved performance over the 7-day recovery period similar to both paws in sham-treated rats. However, when the Aβ toxicity was combined with the endothelin-induced infarcts, both paws showed significant decreases in pellets eaten on the first day of re-testing. This observation was likely caused by combination of a deficit in motor control attributable to the focal cerebral ischemia (contralateral paw) and a possible learning deficit induced by the Aβ toxicity (ipsilateral paw). Furthermore, in the combined Aβ/endothelin-treated rats, there was limited improvement in both paws.
In summary, acute injections of Aβ(25–35) showed increased pathological responses and inflammatory responses throughout the forebrain. We also were able to co-localize an inflammatory response to congophilic Aβ deposition. Our rat model of cerebral ischemia, based on unilateral striatal endothelin injections, demonstrated increases in molecules typically seen in AD pathology, as well as increases in inflammatory cytokines and reactive gliosis. The combination of Aβ toxicity and cerebral ischemia resulted in enhanced of levels of gliosis, particularly in the hippocampus. The focal cerebral ischemia caused a deficit in the contralateral paw and deficits in both paws when Aβ(25–35) was added. This suggests that Aβ toxicity may impair memory and learning associated with the compensating response in the ischemic rats. It remains to be determined if inhibition of the reactive gliosis and/or inflammatory reactions in the brain of theses rats can prevent the cognitive deficits we have observed.
We gratefully thank Lisa Tichenoff, Guanliang Cheng, and Melannie Josseau for their technical assistance. Part of this work was made possible by a Discovery Grant from the Natural Sciences and Engineering Reserach Council of Canada to J.A. Kierman. This study was also partially funded by Grupo Uriach, Poligono Industrial Riera de Caldes, Avinguda Cami Reial, Palau-solita i Plegamans (Barcelona), Spain.
D.F.C is a Career Investigator with Heart and Stroke Foundation of Canada. S.N.W is a Natural Science and Engineering Research Council Scholar.
- Received June 22, 2004.
- Revision received August 27, 2004.
- Accepted September 17, 2004.
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