Interaction Between a Rat Model of Cerebral Ischemia and β-Amyloid Toxicity
II. Effects of Triflusal
Background and Purpose— Clinical data suggest that Alzheimer disease (AD) and stroke together potentiate cognitive impairment. Our rat model demonstrates that this interaction may be mediated through inflammatory cells and pathways. Thus, anti-inflammatory agents such as Triflusal, a nonsteroidal anti-inflammatory agent (NSAID), may provide neuroprotection for susceptible neurons in AD and cerebral ischemia.
Methods— AD was modeled by cerebroventricular injections of β-amyloid (Aβ25–35) and subcortical lacunar infarcts by striatal endothelin injections. Inflammatory mechanisms were examined by immunohistochemical analysis. Behavioral tasks were assessed with the Montoya staircase test.
Results— Triflusal reduced pathologic and inflammatory markers and functional deficits in rats receiving Aβ or endothelin alone but was less effective in the more severe pathology of the combined Aβ/endothelin model.
Conclusions— Higher doses or more prolonged treatment with NSAIDs may be required for more effective neuroprotection in combined AD and stroke conditions.
Clinical data indicate a link between cerebral ischemia and Alzheimer’s disease (AD). Patients with autopsy evidence of AD pathology and lacunar infarcts in the striatum had more cognitive impairment than did patients with AD pathology alone,1,2 suggesting that dementia in AD patients may be worsened with cerebral ischemia.
Inflammatory responses common in both AD and cerebral ischemia include astrocytosis, microgliosis,3 and upregulation of inflammatory cytokines and nuclear factor (NF)-κB.4 Previously, rat models of cerebral ischemia or β-amyloid (Aβ) induced AD-like pathology and produced inflammatory responses.5 However, the combination of these 2 models elicited an even stronger inflammatory response.
Anti-inflammatory agents such as nonsteroidal anti-inflammatory agents (NSAIDs) may prove to be potential therapeutic agents in AD and cerebral ischemia and may be particularly effective in inhibiting the enhanced neuroinflammation and dementia when both AD and cerebral ischemia are present.
Triflusal (2-acetoxy-4-trifluoromethylbenzoic acid) is an antiplatelet agent structurally related to aspirin, with important differences in pharmacological, biochemical, and clinical aspects.6 Triflusal is rapidly transformed to its main metabolite 3-hydroxy-4-trifluoromethylbenzoic acid (HTB) after oral administration in humans.6 In major clinical trials, Triflusal has demonstrated efficacy similar to aspirin in the secondary prevention of ischemic episodes but with a markedly reduced risk of cerebral and systemic hemorrhage.7
Recently, it has been demonstrated that Triflusal and HTB are potent in vitro and in vivo inhibitors of the activation of NF-κB and NF-κB–regulated genes. Furthermore, orally administered Triflusal is neuroprotective and downregulates NF-κB, inflammatory mediators, and microglial activation in an excitotoxic (N-methyl-d-aspartate) injury model in the postnatal rat brain.8,9
A combined rat model of AD and cerebral ischemia describing exacerbated levels of inflammation and behavioral deficits has recently been established.5 The purpose of this study was to examine the effects of anti-inflammatory treatment on these models. To examine the therapeutic potential of Triflusal experimentally, AD pathology was generated with an intracerebroventricular (ICV) injections of Aβ(25–35), the toxic fragment of Aβ, alone or in combination with striatal injections of the potent vasoconstrictor endothelin to mimic the subcortical lacunar infarcts seen clinically.10
Materials and Methods
All experimental procedures were carried out according to the animal care guidelines of the University of Western Ontario. Male Wistar rats (250 to 300 g) were divided into 4 groups: ICV injections of Aβ(25–35) (Bachem); 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 with 40 mg/kg IP pentobarbital (Somnotol). Body temperature was maintained at 37°C. Aβ(25–35) (50 nmol in 10 μL of saline) was injected bilaterally into the lateral ventricles. For details on Aβ(25–35) composition, preparation, and administration see Sigurdsson et al10 and Whitehead et al.5 Two endothelin injections (6 pmol in 3 μL of saline), 1 mm apart, were made into the right striatum.5,11 Sham procedures involved all of the surgical steps without injections of Aβ(25–35) or endothelin. After wound suturing, all rats received 40 mg/kg IM buprenorphine and were allowed to recover from surgery for 7 days.
After surgery and recovery, mice received either oral administration of 30 mg/kg Triflusal or vehicle via oral gavage for 8 days (including the day of surgery). For details on vehicle and Triflusal preparation, see Bayon et al.12
Twenty-one days after surgery, all animals were humanely killed with a pentobarbital overdose and perfused transaortically, first with saline and then with 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 with a cryostat.
For detailed procedures, see Whitehead et al.5 Additionally, cyclooxygenase (COX)-2 mouse monoclonal (1:1000, Fitzgerald Industries International) and cleaved Cas-3 rabbit polyclonal (1:500, Cell Signaling Technology,) primary antibodies were used.
For a detailed protocol, see Whitehead et al5
Digital photographs were taken with a light microscope (Leitz Diaplan). An investigator was blinded to the identification of the rat sections while analysis was being performed. All immunohistochemical data to be compared were processed at the same time with the same solutions to reduce any nonexperimental variation in intensity. A grading scale was based on both observable size and intensity of staining. Average optical densitometry measurements were calculated from 3 hippocampal sections. Measured areas were consistent between animals. Numbers of amyloid precursor protein (APP) accumulations were calculated in the cortex of each animal (average number of sections measured=20). Statistical analysis with ANOVA and Tukey’s post hoc test with a significance level of P<0.05 (n=4 for each group) was done for immunohistochemical data. Statistical analysis with Student’s t test was performed on the staircase test data with a significance level of P<0.05 (n=10 for each group).
As previously demonstrated,5 endothelin induced increases in inflammatory, apoptotic, and AD-associated pathologic markers in the ipsilateral striatum and thalamus (Figure 1A, 1C, 1E, 1G, and 1I; Table⇓). Triflusal treatment resulted in a strong reduction in APP (Figure 1B), Tau-2 (Figure 1D), OX-6 (Figure 1J), and COX-2 (Table⇓) immunostaining; a mild reduction in NF-κB (p65) and tumor necrosis factor (TNF)-α (Table 1⇓) immunostaining; and no reduction in glial fibrillary acidic protein (GFAP) immunostaining (Figure 1F).
In the cortex, Aβ(25–35) injections elicited increases in many proteins (Table⇑), including cellular and accumulation-type (encompasses >1 cell and stains both intracellular and extracellular components) APP staining (Figure 2A), cellular Tau-2 (Figure 2C), and positive accumulation-type and cellular COX-2 (Figure 2E). Triflusal treatment greatly reduced APP (Figure 2B), Tau-2 (Figure 2D), and COX-2 (Figure 2F) immunostaining in the cortex. Triflusal also reduced OX-6, NF-κB (p65), and TNF-α immunostaining in the cortex (Table⇑). Triflusal had no effect on GFAP-labeled astrocytes in the cortex (Table⇑).
Aβ(25–35) injections resulted in increased immunostaining in the corpus callosum (Table⇑), including somatic and axonal APP (Figure 2G) cellular NF-κB (p65) (Figure 2I), and GFAP-immunostained astrocytes (Figure 2K). Triflusal treatment resulted in a strong reduction in APP (Figure 2H), NF-κB (p65) (Figure 2I), GFAP-labeled astrocytes (Figure 2L), OX-6, COX-2, Tau-2, and TNF-α (Table⇑) in the corpus callosum.
In the hippocampus, Aβ(25–35) induced increases in Tau-2 immunostaining in the CA2/CA3 regions of the hippocampus (Figure 3A) and GFAP-labeled astrocytes in the CA1–CA3 regions (Figure 3C). Aβ(25–35) increased OX-6–labeled microglia (Figure 3E) and TNF-α (Figure 3G) immunostaining in the thalamus. Triflusal treatment resulted in a reduction in Tau-2 (Figure 3B), OX-6, APP, NF-κB (p65), and COX-2 (Table⇑), with no reduction of GFAP immunostaining in the hippocampus (Figure 3D). Triflusal reduced OX-6–labeled microglia (Figure 3F) and TNF-α immunostaining in the thalamus (Figure 3H) and reduced APP, NF-κB (p65), and TNF-α immunostaining in the hypothalamus (Table⇑).
Combined Aβ/endothelin administration resulted in large increases in inflammatory, apoptotic, and AD-related pathologic markers in numerous brain regions (Figure 4A, 4C, 4E, 4G, 4I, and 4K; Table⇑). Triflusal treatment resulted in a reduction in Cas-3 immunostaining in the striatum, corresponding to the area of ischemic damage (Figure 4B), OX-6 immunostaining in the striatum posterior to the endothelin injection site (Figure 4F), and OX-6–labeled microglia in the corpus callosum posterior to the Aβ injection site (Figure 4L).
Some immunohistochemical results that were quantified by optical densitometry are shown in Table 1B, indicating that combined treatment elicits significant increases of immunostaining compared with Aβ or endothelin alone. Triflusal significantly reduced select immunostaining in Aβ- or endothelin-alone–treated rats but did not significantly decrease immunostaining in combination-treated rats, except for OX-6 in the right hippocampus.
At the start of retesting, 7 days after surgery (day 8), Aβ-treated rats that received vehicle showed a significant decrease in the number of food pellets eaten (−5.4±1.4) compared with day −1, which was prevented by Triflusal treatment (Figure 5). Combined Aβ/endothelin-treated rats showed significant decreases in pellets eaten on days 8 through 12 compared with day −1, although this was not affected by treatment with Triflusal (Figure 5).
Individual paw performance was calculated as a percent change. Endothelin-treated rats treated with vehicle had significant impairment in the left paw (contralateral to the stroke) on day, 8 (75±13% of the pretreatment average), 9, 11, and 15 but only on day 8 after treatment with Triflusal (Figure 6). Aβ-treated rats that received vehicle showed a deficit in the right paw only on day 8 (67±10% of the pretreatment average), which was prevented by Triflusal. Combined Aβ/endothelin-treated rats that received vehicle had deficits throughout the postsurgery testing period in both the left and right paws. With Triflusal treatment, the right and left paws recovered by days 9 and 12, respectively (Figure 6).
Our results strongly suggest that the anti-inflammatory action of Triflusal is an effective mechanism of neuroprotection. Previous studies have shown that Triflusal treatment reduces NF-κB activation and glial reactivity, as well as inducible nitric oxide synthase, COX-2, interleukin-1β, and TNF-α expression, after an excitotoxic lesion in the postnatal rat brain.8,9
The focal-ischemia model with endothelin injections into the striatum was chosen to mimic the size and location of the multiple infarcts that are significantly correlated with increases in cognitive deficits in patients with evidence of Alzheimer-type lesions.1 As we have demonstrated here and previously,5,11 this model in the rat appears to be an effective way of creating ischemia with a relatively low level of inflammation, compared with the larger insults obtained with middle cerebral artery occlusion. Thus, this ischemic model, with relatively low levels of inflammation, allows for the assessment of its interaction with the inflammatory responses produced by Aβ toxicity. Furthermore, the bilateral ICV injections of Aβ(25–35) serve as an effective rat model to produce some of the pathologic and behavioral changes of AD.5,13,14
In the present investigation, bilateral ICV injections of Aβ(25–35) and/or unilateral striatal injections of endothelin produced pathologic and behavioral results similar to those previously described.5 Rats receiving endothelin alone without any treatment had increases in inflammatory, apoptotic, and pathologic correlates of AD in the striatum. Aβ-injected rats showed AD-like pathologic and inflammatory responses in several areas of the brain, including the cortex, corpus callosum, hippocampus, thalamus, and hypothalamus. In addition, as previously demonstrated,5 the combination of Aβ and endothelin injections produced greater functional deficits and a greater inflammatory and pathological response, particularly in the hippocampus, than either Aβ or endothelin alone.
Generally, Triflusal showed neuroprotective effects in rats receiving endothelin. Triflusal strongly reduced APP, Tau-2, and COX-2 immunostaining; and reduced the spread of immunostaining of OX-6–positive microglia to the posterior striatum. Previously, in vitro studies have shown that aspirin can protect neurons from hypoxia by mechanisms such as inhibition of the excitatory amino acid release, inhibition of inducible nitric oxide synthase expression, or the delay of intracellular ATP loss.15–17 Furthermore, aspirin inhibits NF-κB translocation, and this is the mechanism by which aspirin is neuroprotective in vitro.18–20 Aspirin has also been shown to be neuroprotective in vivo in animal models of stroke.15–17 Triflusal appears to have a similar therapeutic potential as aspirin, but unlike aspirin, selectively inhibits COX-2. Our immunohistochemical results suggest that the anti-inflammatory action of Triflusal may be the mechanism of neuroprotection.
This is the first study examining the role of Triflusal as an anti-inflammatory agent in an experimental model of AD in the rat. Triflusal, in general, reduced levels of pathologic and inflammatory correlates of AD throughout the brain of rats receiving Aβ(25–35) injections. The anti-inflammatory drug ibuprofen has also been shown to decrease interleukin-1β and GFAP-positive astrocytes in a mouse model of AD.21 Furthermore, epidemiologic studies have suggested that prophylactic treatment with anti-inflammatory agents decrease the risk of developing AD.22 Therefore, Triflusal may, by reducing the severity of inflammation caused by Aβ toxicity in the rat, be beneficial in humans in reducing the risk of AD.
The combination of endothelin and Aβ injections in the rat elicited enhanced pathologic and inflammatory responses. Although Triflusal was able to reduce some of the inflammatory signals, the degree of reduction was not as effective as seen in rats administered Aβ or endothelin alone. It is possible that the neuroprotective action of Triflusal was unable to ameliorate the much greater inflammatory response attributable to the combination of Aβ toxicity.
The functional data obtained with the Montoya staircase apparatus appear to support the changes observed with the pathologic and inflammatory markers. Aβ injections, with and without endothelin, impaired performance after surgery. The increases in GFAP and OX-6 in the hippocampus after Aβ administration may be correlated to the behavioral deficit. There was a greater degree of impairment in combined Aβ/endothelin injections compared with Aβ alone, and the duration of recovery was also longer. Furthermore, the combination of Aβ/endothelin was also concomitant with greater increases in immunohistochemical pathologic markers in the hippocampus and cortex.
Treatment with Triflusal eliminated the behavioral deficit seen in rats receiving Aβ alone. However, Triflusal treatment did affect the behavioral deficit of combined Aβ/endothelin in rats. These behavioral results with Triflusal suggest that the pathologic response to the combination of Aβ/endothelin was too great for the neuroprotective action of Triflusal and thus, was unable to rescue functional deficits seen in the staircase test.
As previously demonstrated,5 endothelin-treated rats have a prolonged deficit in the paw contralateral to the infarct, whereas the ipsilateral paw improves over time. With Triflusal treatment, there was only a transient 1-day impairment in the contralateral paw, with no significant difference between the 2 paws at the end of the retesting period, suggesting a positive functional outcome with drug treatment. Combined Aβ/endothelin-treated rats had prolonged impairment in both paws that was not affected by treatment with Triflusal.
In summary, Triflusal inhibits inflammation induced by endothelin-derived cerebral ischemia in the rat. Both AD pathologic and inflammatory outcomes of Aβ toxicity were also inhibited by Triflusal in the rat. Functional deficits, as measured by the staircase test, could be correlated to the changes seen in Aβ- and endothelin-induced pathology and inflammation. The anti-inflammatory effects of Triflusal may account for observed behavioral improvement in the stroke or AD model but had no effect in the combined model.
- Received April 22, 2005.
- Accepted May 2, 2005.
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