Published online before print February 26, 2009, doi: 10.1161/STROKEAHA.108.531509
(Stroke. 2009;40:1482.)
© 2009 American Heart Association, Inc.
Original Contributions |
Angiotensin AT2 Receptor Stimulation Causes Neuroprotection in a Conscious Rat Model of Stroke
From the Department of Pharmacology (C.A.M., A.V., R.E.W.), Monash University, Clayton, Victoria, Australia; and the Department of Pharmacology (J.K.C.), University of Melbourne, Parkville, Victoria, Australia.
Correspondence to Robert E. Widdop, PhD, Department of Pharmacology, Monash University, Clayton Victoria 3800, Australia. E-mail robert.widdop{at}med.monash.edu.au
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferencesSurgical ProceduresTreatmentsSystolic Blood PressureStroke InductionAssessment of Motor FunctionQuantification of Ischemic...Neuronal ImmunohistochemistryLocalization of Superoxide by...Angiotensin 2 Receptor...Statistical Analysis |
|---|
Background and Purpose— The angiotensin II type 2 receptor (AT2R) is implicated to be neuroprotective in stroke, although this premise has not been directly tested. Therefore, we have examined the neuroprotective effect of AT2R stimulation after intracerebroventricular administration of AT2R agonist CGP42112 in a conscious rat model of stroke.
Methods— Spontaneously hypertensive rats were treated with either CGP42112 (0.1 to 10 ng/kg/min intracerebroventricularly) alone or in combination with the AT2R antagonist PD123319 (36 ng/kg/min intracerebroventricularly) beginning 5 days before stroke induction. A focal reperfusion model of stroke was induced in conscious spontaneously hypertensive rats by administering endothelin-1 to the middle cerebral artery through a surgically implanted cannula. Behavioral tests were used to assess the severity of neurological deficit as a result of the ischemic event. Cortical and striatal infarct volumes were measured 72 hours poststroke.
Results— Blood pressure was unaffected by treatments. CGP42112 dose-dependently reduced cortical infarct volume poststroke, and PD123319 abolished the neuroprotective effect of CGP42112. PD123319 had no effect on infarct volume alone. These results were consistent with the behavioral findings, indicating that CGP42112 reduced motor deficit on the ledged beam test at 72 hours poststroke and immunohistochemical analyses showing that CGP42112 increased neuronal survival and minimized the loss of AT2R expression in the infarcted region.
Conclusion— Based on infarct, behavioral, and immunohistochemical data, these results indicate that centrally administered CGP42112 exhibits a neuroprotective effect, which was independent of blood pressure. Thus, for the first time, we have shown that central AT2R stimulation is neuroprotective in a conscious rat model of stroke.
Key Words: AT2receptors • cerebral infarction • cerebral ischemia • neuroprotection • SHR • stroke
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferencesSurgical ProceduresTreatmentsSystolic Blood PressureStroke InductionAssessment of Motor FunctionQuantification of Ischemic...Neuronal ImmunohistochemistryLocalization of Superoxide by...Angiotensin 2 Receptor...Statistical Analysis |
|---|
Angiotensin II binds to either the angiotensin II type 1 receptor (AT1R), which is responsible for most of the physiological and pathological actions of angiotensin II, or the angiotensin II type 2 receptor (AT2R), which opposes the actions of the AT1R. AT1R antagonists are commonly used in the treatment of hypertension, one of the main risk factors for stroke.1 Over the past decade, it has become apparent that the beneficial effects of AT1R antagonists may extend beyond the control of blood pressure.2,3
Animal studies have convincingly demonstrated that, even if given during the reperfusion stage of stroke, AT1R antagonists improve cerebral perfusion during an ischemic event, thus reducing neuronal damage4 possibly by increasing cerebral vascular compliance, resulting in increased cerebrovascular perfusion during stroke.5,6 In this context, treatment with an AT1R antagonist per se caused an increase in vascular AT2R in the cerebral circulation.7 In addition to their beneficial local vasodilator effects, it is known that sartan compounds exhibit antiapoptotic, anti-inflammatory, and antioxidant properties, indicating that blockade of the AT1R may have both vascular-dependent and -independent neuroprotective actions in the setting of stroke.8–11 Integral to the neuroprotective role of the AT2R is its role in neuronal regeneration. Several models of nerve injury have elegantly shown that the AT2R has regenerative capabilities associated with restored behavioral function and anatomic innervation after sciatic nerve crush and optical axotomy as well as increased neurite extension in cell culture.12–16
It has been speculated that the increased stimulation of the AT2R may be responsible for some of the therapeutic effects observed during AT1R blockade.17,18 In this context, coadministration of an AT2R antagonist negated neuroanatomical and neurological benefit of candesartan or irbesartan in the filament and cerebral artery occlusion stroke models, demonstrating that AT2R activation contributed to the effect of AT1R antagonists.12,19 Other indirect evidence that AT2R stimulation is neuroprotective in the setting of stroke is that AT2R-deficient mice have larger infarct volumes and poorer neurological outcome after stroke.20,21 Moreover, AT1R antagonists are less effective in AT2R-deficient mice, again confirming that AT2R play a pivotal role in the effect of AT1R antagonists.21
However, to date, all investigations examining the neuroprotective nature of the AT2R as recently reviewed by Thone-Reineke et al22 have been indirect because they invoke a diminished or reversible sartan effect during genetic or pharmacological AT2R blockade. Therefore, the main aim of the present study was to stimulate directly AT2R because we hypothesized that stimulation of the central AT2R would be neuroprotective in its own right in a conscious rat model of stroke, thus representing a new pharmacological target in stroke.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferencesSurgical ProceduresTreatmentsSystolic Blood PressureStroke InductionAssessment of Motor FunctionQuantification of Ischemic...Neuronal ImmunohistochemistryLocalization of Superoxide by...Angiotensin 2 Receptor...Statistical Analysis |
|---|
Full details can be found in the supplement material, available online at http://stroke.ahajournals.org.
Surgical Procedures
Male 15- to 16-week-old (270 to 320 g) spontaneously hypertensive rats (SHR) had a 23-gauge stainless steel guide cannula stereotaxically implanted into the piriform cortex 2 mm dorsal to the right middle cerebral artery as previously described.23,24 The stereotaxic coordinates were modified for this strain of rat (0.2 mm anterior, –4.7 mm lateral, and –7 mm ventral relative to bregma). An additional 23-gauge intracerebroventricular (ICV) cannula was also stereotaxically implanted into the left lateral ventricle (–0.8 mm anterior, +1.5 mm lateral, and –3.2 mm ventral relative to bregma) for drug administration. These studies were approved by a Monash University Animal Ethics Committee.
Treatments
Rats were randomly allocated to the following treatment groups: 0.1 (n=6), 1 (n=9), or 10 ng/kg/min CGP42112 (n=6; gift from Novatis); CGP42112 (1 ng/kg/min)+PD123319 (36 ng/kg/min; n=10; gift from Pfizer); PD123319 (36 ng/kg/min) alone (n=10), or saline (n=11). The ICV cannula and osmotic minipump containing these treatments were implanted 5 days before stroke and continued to deliver treatment for 3 days poststroke. Two additional sham groups, with or without CGP42112 1 ng/kg/min, underwent surgery without stroke induction. These sham groups acted as surgical and histological controls, respectively.
Systolic Blood Pressure
Systolic blood pressure (SBP) was measured before the start of ICV treatment and also 72 hours poststroke (Day 14).
Stroke Induction
Stroke was induced in conscious animals by titrating the administration of the vasoconstrictor, ET-1 (Auspep), to cause temporary occlusion of the right middle cerebral artery.23,24 ET-1 was infused through a 30-gauge injector protruding 3 mm beyond the end of the previously implanted guide cannula. Stroke was characterized by behavioral indicators24,25 modified to be specific for the SHR strain. Typical behaviors that were observed were continuous contralateral or ipsilateral circling behavior; clenching, dragging, or failure to extend the forelimb contralateral to the side of ET-1 infusion; and forepaw shuffling, jaw flexing, or chin rubbing while circling. Each stroke was graded based on these predetermined behavioral changes using a scale of 1 to 4 with 1 being a mild stroke and 4 being a severe stroke. Core body temperature was monitored rectally using a digital thermometer over a 3-hour period. The experimenter was blinded to the treatments. Only rats with a stroke grade of 4, exhibiting at least 7 of the previously mentioned behaviors, were used for the purpose of this investigation, and any animal with a core temperature >40°C after stroke was euthanized.
Assessment of Motor Function
Motor coordination and limb function were assessed using the ledged beam as previously described.26 The ledged beam test was conducted before treatment (Day 0), after recovery from cannula implantation (Day 6), immediately before stroke induction (Day 11), 24 hours poststroke induction (Day 12), and 72 hours poststroke induction (Day 14). All values were compared with presurgery; therefore, each rat acted as its own control. The number of total foot steps taken to run the length of the beam was recorded as well as the number of steps taken on the ledge by each foot. The latter number was recorded as a percentage of the total number of footsteps taken and recorded as percentage error.
Quantification of Ischemic Damage
At 72 hours after stroke, rats were reanesthetized and perfused (25 mL/min) with physiologically buffered saline (0.1 mol/L; pH 7.4) through the apex of the heart. Brains were removed, frozen over liquid nitrogen, and stored at –80°C. Coronal cryostat sections (16 µm) were cut at 8 predetermined levels of the forebrain (–3.20 mm to 6.80 mm relative to bregma), slide-mounted, and stored at –80°C. The infarct area and volume were calculated using a method developed by Callaway and colleagues.27 The infarct volume was quantified by integrating the area of damage with the distance between each level.28
Neuronal Immunohistochemistry
Neuron integrity was assessed using the neuronal marker NeuN. The number of NeuN-positive cells (identified by anti-NeuN IgG antibody) was counted within a 1-mm2 site in the infarcted and noninfarcted region of the ipsilateral hemisphere and 2 matched regions of the contralateral hemisphere.
Localization of Superoxide by Dihydroethidium Staining
Dihydroethidium (2 µmol/L; Invitrogen) was used as previously described29 to detect superoxide in frozen coronal cryostat sections (16 µm; –0.8 mm relative to bregma in vehicle).
Angiotensin 2 Receptor Immunohistochemistry
AT2R expression was assessed as previously described by Roulston et al.30 Images were taken from the cortical noninfarcted and infarcted regions of the ipsilateral hemisphere and 2 matched regions of the contralateral hemisphere. The number of cells immunopositive for AT2R was counted within a 0.25-mm2 site for each region.
Statistical Analysis
Results are presented as mean±SEM. Ledged beam and blood pressure data were analyzed using 2-way repeated-measures analysis of variance (ANOVA) with individual differences assessed using Bonferroni posttest. Infarct area, AT2R expression, neuronal expression, and superoxide production were assessed using one-way ANOVA with individual differences assessed using Dunnett’s posttest. All statistical analyses were performed using PRISM (GraphPad),and a value of P<0.05 was considered statistically significant.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferencesSurgical ProceduresTreatmentsSystolic Blood PressureStroke InductionAssessment of Motor FunctionQuantification of Ischemic...Neuronal ImmunohistochemistryLocalization of Superoxide by...Angiotensin 2 Receptor...Statistical Analysis |
|---|
Systolic Blood Pressure
There was no significant difference in SBP between any of the treatment groups, and CGP42112 did not significantly alter SBP (Table).
|
Infarct Volume
When compared with vehicle (112.60±21.03 mm3), CGP42112 (1 ng and 10 ng/kg/min) significantly reduced cortical infarct volume at 72 hours poststroke (16.43±5.21 and 9.34±5.30 mm3, respectively; P<0.01, P<0.001 versus vehicle). In addition, CGP42112 (10 ng/kg/min) also reduced striatal infarct volume when compared with vehicle (12.40±3.66 and 30.27±4.58 mm3, respectively; P<0.05). While the lowest dose of CGP42112 also reduced cortical damage, this result did not reach statistical significance. This neuroprotective effect observed in the animals receiving CGP42112 (1 ng/kg/min) was reversed by PD123319; however, PD123319 had no effect when administered alone (Figure 1).
|
Motor Function
Stroked rats treated with vehicle showed a significant increase in percentage errors on the ledged beam at 1 and 3 days poststroke indicating that the test is sensitive to stroke-induced deficit. Importantly, there was no change in the percentage errors in the sham-operated, nonstroked animals, confirming that any deficit observed was a stroke-related injury and not due to surgical procedures. CGP42112 (1 and 10 ng/kg/min) significantly reduced errors 1 day poststroke (12±11% and 13±7%, respectively) when compared with vehicle (68±12%; P<0.05 versus vehicle). Furthermore, at 3 days poststroke, all doses of CGP42112 significantly reduced errors (22±16%, 5±3%, and 10±10% for 0.1, 1 and 10 ng/kg/min, respectively) versus vehicle (73±9%; P<0.05 to 0.01). PD123319 negated the improvements in performance observed in CGP42112-treated rats while having no effect when administered alone. There was no change in percentage error in the sham group at any of the times tested (Figure 2).
|
Neuronal Expression
The number of neurons as indicated by NeuN-immunopositive labeling was significantly lower in the cortical infarct region of the ipsilateral hemisphere in vehicle-treated rats after stroke (8.14±4.10 NeuN-positive cells per mm2) when compared with the nonstroked, sham-operated animals (220.00±32.90 NeuN-positive cells per mm2; P<0.05; Figure 3). The loss of neuronal expression in the vehicle-treated group extended to the matched region corresponding to the infarct region on the contralateral hemisphere. Importantly, CGP42112 (1 ng/kg/min) ameliorated the loss of NeuN-immunopositive cells (82.75±40.17 NeuN-positive cells per mm2) in the ipsilateral infarcted region. Coadministration of PD123319 with CGP42112 reversed the CGP42112-induced increase in neuronal survival in the ipsilateral hemisphere. PD123319 itself did not alter neuron number compared with vehicle-treated stroked rats.
|
Superoxide Production
Superoxide production was elevated globally after stroke induction except for in the infarcted region of the ipsilateral hemisphere of the CGP42112 (1 ng/kg/min) -treated group. There was no treatment effect on superoxide production on the contralateral hemisphere or in the noninfarcted region (Figure 4).
|
AT2 Receptor Immunohistochemistry
AT2R expression was lower in the cortical infarct region in the ipsilateral hemisphere of stroked vehicle-treated rats. CGP42112 (1 ng/kg/min) reversed the loss of AT2R expression in the infarct region (Figure 5). Additionally, CGP42112 tended to increase AT2R expression in the noninfarcted region of the ipsilateral hemisphere as well as in the contralateral hemisphere. In the absence of stroke, CGP42112 treatment had no effect on the number of AT2R-immunopositive cells (data not shown). There was no significant change in the number of AT2R-immunopositive cells in the striatal region (data not shown).
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferencesSurgical ProceduresTreatmentsSystolic Blood PressureStroke InductionAssessment of Motor FunctionQuantification of Ischemic...Neuronal ImmunohistochemistryLocalization of Superoxide by...Angiotensin 2 Receptor...Statistical Analysis |
|---|
This investigation is the first study to show directly that stimulation of the AT2R is neuroprotective against an ischemic stroke. Using a model of stroke in conscious SHRs,23,24 we demonstrated that pretreatment with the AT2R agonist CGP42112, across a nanomolar dose range, almost completely prevented cortical infarction and preserved brain function assessed by neuroanatomical and behavioral measurements, all of which was independent of changes in SBP.
Previous studies have indirectly implicated the beneficial effect of AT2R stimulation after stroke, since the reduction in infarct volume and neurological deficit observed during AT1R blockade were reversed with the coadministration of an AT2R antagonist.12,19,22 Furthermore, when compared with wild-type mice, AT2R knockout mice have larger infarct areas and poorer neurological outcome after stroke.20 Thus, to date, all findings regarding the role of the AT2R have been based on indirect studies inhibiting AT2R function by pharmacological or genetic means. Moreover, many of the previous studies use models of stroke that induce large infarcts, which are less likely to occur in a clinical setting and/or permanently occlude the middle cerebral artery, which eliminates the potentially damaging reperfusion phase of stroke.12,19
To this end, we examined the role of the AT2R in a model of stroke that has several advantages over other models. First, rats were hypertensive, an important risk factor for stroke. Second, animals were conscious during the stroke procedure, thus avoiding the influence of anesthesia, which itself can cause neuroprotection. Third, by using a vasoconstrictor such as ET-1, the current model exhibits initial cerebral vasoconstriction, reducing perfusion, followed by a reperfusion phase during ET-1 washout. Moreover, the severity of stroke can be controlled using a predetermined scale of behavioral indicators, which correlates with the degree of neuronal damage in this model.25
The current results demonstrated a dose-related neuroprotective effect of CGP42112 that was more striking in the cortex than in the striatum. Consistent with our findings, it was previously reported, using this model, that there was less neuroprotection in the striatal region when compared with the cortical region, because the striatal region is generally considered to be the core of the infarct and refractory to therapeutic intervention.24,31
These results of the current study are remarkable for several reasons. Often, the effects of AT2R activation need to be unmasked after AT1R blockade, as is the case of the peripheral vasodilator effects of CGP42112 in conscious SHRs.32,33 However, we found that the AT2R agonist alone had a striking effect on infarct volume even in the absence of AT1R blockade and indeed was comparable in its neuroprotective effect to that of an AT1R antagonist in the only study that has centrally administered candesartan, albeit in a different model.12 Furthermore, the doses of CGP42112 were at least 1000-fold less than what we have previously used for examining vasodilatation after intravenous administration.32,33 Such differences point toward increased sensitivity to the functional effects of central AT2R activation. However, we found that the AT2R had no tonic influence, because the AT2R antagonist PD123319 had no effect on stroke outcome when administered alone, although when coadministered with CGP42112, PD123319 negated any neuroprotective effect, thus confirming that CGP42112 was acting via the AT2R. Importantly, these effects occurred in the absence of a reduction in SBP, which is well known to be neuroprotective, although it is possible that local cerebral vasodilatation via AT2R stimulation may play a beneficial role.4–7
Our behavioral data indicated that all 3 doses of CGP42112 reduced motor deficit at 24 hours poststroke, although there was some variation in responses at this time, which may reflect the individual variation often seen in behavioral studies and the relatively small sample size. In any case, this neuroprotective effect was more striking after 72 hours. Moreover, in the current study, the lowest dose of CGP42112 improved motor performance after stroke, whereas the reduction in infarct volume failed to reach statistical significance. AT2R signaling has been associated with many neuron-regenerative processes, including increased dendritic branching and neurite outgrowth,12,15 thus facilitating functional recovery. Importantly, the behavioral improvement mediated by CGP42112 was reversed by the coadministration of an AT2R antagonist, again suggesting that the neuroprotection was mediated via AT2R. Consistent with this notion, stroke resulted in a complete loss of neurons identified by the neuronal marker NeuN12 in the infarcted tissue compared with the sham group, and AT2R stimulation significantly increased neuronal survival in a PD123319-sensitive manner. CGP42112 has also been reported to bind to non-angiotensin II sites on inflammatory cells34,35; however, this mechanism is not likely to play a major role in the current study because PD123319 reversed all of the neuroprotective effects of CGP42112 in the current study.
Previous research has shown that AT2R are neuronally expressed12 and that there is a time-dependant modulation in AT2R expression after stroke.36 In the present study, the loss of the AT2R in the infarct region is also most likely to be a consequence of a loss of neuronal cells in the ischemic area, which has been confirmed by NeuN staining. CGP42112 increases neuronal survival and therefore also preserves AT2R expression specifically in the ischemic area. It is interesting to note that some loss of neuronal cells in the vehicle-treated group also extends to the matched region on the contralateral noninfarcted hemisphere. The mechanism through which this occurs is presently unclear; however, previous findings have indicated significant changes in areas remote from the ischemic lesion.37,38 CGP42112 did not alter AT2R in the absence of stroke, confirming that central AT2R changes are most likely secondary to neuronal damage.
Oxidative stress is a major contributing factor to the pathology of stroke. AT1R stimulation causes an increase in superoxide production, whereas AT2R opposes this effect because AT2R-deficient mice had elevated levels of superoxide in the ischemic area after stroke.20,39 In the current study, stroke caused a global increase in superoxide production, and this effect was attenuated in the infarct region by CGP42112, ie, in the same area in which AT2R expression has been preserved. While the role of the AT2R in oxidative stress is still not fully understood, the inverse relationship between superoxide production and AT2R expression suggests that the AT2R reduces oxidative stress induced during ischemia. In addition, superoxide production was increased on the contralateral noninfarcted hemisphere suggesting that stroke is a global phenomenon. In accordance, using the same model, Miller et al38 found that superoxide production was increased in the contralateral cerebral vessels at 24 hours poststroke.
In conclusion, we have directly stimulated AT2R using CGP42112 in a clinically relevant model of stroke and have demonstrated a striking reduction in both cortical and striatal infarct volume. These findings are consistent with markedly improved behavioral outcome indicted by the striking reduction in motor deficit at both 24 hours and 72 hours poststroke. We confirmed that CGP42112 was acting through the AT2R by reversing all the neuroprotective changes with the coadministration of PD123319. Neuroprotective mechanisms are likely to involve AT2R-mediated increased neuronal survival in addition to a well-documented AT2R-mediated vasodilator role, leading to preservation of neuronal function and AT2 receptor expression while having a small inhibitory effect on superoxide production. These studies provide proof-of-principle for a role of AT2R stimulation in neuroprotection, although a limitation of the present study was that CGP42112 was given as a pretreatment before stroke induction. Clearly, studies are required in which CGP42112 is given after stroke to more closely mimic the clinical situation. Collectively, these findings highlight the AT2R as a potential therapeutic target in the treatment of stroke, particularly using AT2R agonists.
Received July 18, 2008; revision received October 7, 2008; accepted October 17, 2008.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferencesSurgical ProceduresTreatmentsSystolic Blood PressureStroke InductionAssessment of Motor FunctionQuantification of Ischemic...Neuronal ImmunohistochemistryLocalization of Superoxide by...Angiotensin 2 Receptor...Statistical Analysis |
|---|
1. McInnes G. The expanding role angiotensin receptors blockers in the management of the elderly hypertensive. Curr Med Res Opin. 2003; 19: 452–453.[CrossRef][Medline] [Order article via Infotrieve]
2. Chapman N, Huxley R, Anderson C, Bousser G, Chalmers J, Colman S, Davis S, Donnan G, MacMahon S, Neal B, Warlow C, Woodward M. Effects of a perindopril-based blood pressure-lowering regimen on the risk of recurrent stroke according to stroke subtype and medical history. Stroke. 2004; 35: 116–121.
3. Schrader J, Luders S, Kulschewski A, Berger J, Zidek W, Treib J, Einhaupl K, Diener H, Dominiak P. The ACCESS study. Evaluation of acute candesartan cilexetil therapy in stroke survivors. Stroke. 2003; 34: 1699–1703.
4. Engelhorn T, Goerike S, Doerfler A, Okorn C, Forsting M, Heusch G, Schulz R. The angiotensin II type 1-receptor blocker candesartan increases cerebral blood flow, reduces infarct size, and improves neurological outcome after transient cerebral ischemia in rats. J Cereb Blood Flow Metab. 2004; 24: 467–474.[CrossRef][Medline] [Order article via Infotrieve]
5. Nishimura Y, Ito T, Saavedra J. Angiotensin II AT1 blockade normalizes cerebrovascular autoregulation and reduces cerebral ischemia in spontaneously hypertensive rats. Stroke. 2000; 31: 2478–2486.
6. Ito T, Yamakawa H, Bregonzio C, Terron J, Falcon-Neri A, Saavedra J. Protection against ischemia and improvement of cerebral blood flow in genetically hypertensive rats by chronic pretreatment with an angiotensin II AT1 antagonist. Stroke. 2002; 33: 2297–2303.
7. Zhou J, Pavel J, Macova M, Yu Z, Imboden H, Ge L, Nishioku T, Dou J, Delgiacco E, Saavedra J. AT1 receptor blockade regulates the local angiotensin II system in cerebral microvessels from spontaneously hypertensive rats. Stroke. 2006; 37: 1271–1276.
8. Ando H, Zhou J, Macova M, Imboden H, Saavedra J. Angiotensin II AT1 receptor blockade reverses pathological hypertrophy and inflammation in brain microvessels of spontaneously hypertensive rats. Stroke. 2004; 35: 1726–1731.
9. Dai W, Funk A, Herdegen T, Unger T, Culman J. Blockade of central angiotensin AT1 receptors improves neurological outcome and reduces expression of AP-1 transcription factors after focal brain ischemia in rats. Stroke. 1999; 20: 2391–2399.
10. Lou M, Blume A, Zhao Y, Golhlke P, Deuschl G, Herdegen T, Culman J. Sustained blockade of brain AT1 receptors before and after focal cerebral ischemia alleviates neurological deficits and reduces neuronal injury, apoptosis and inflammatory responses in the rat. J Cereb Blood Flow Metab. 2004; 24: 536–547.[CrossRef][Medline] [Order article via Infotrieve]
11. Zhou J, Ando H, Macova M, Dou J, Saavedra J. Angiotensin II AT1 receptor blockade abolishes brain microvascular inflammation and heat shock protein responses in hypertensive rats. J Cereb Blood Flow Metab. 2005; 25: 878–886.[CrossRef][Medline] [Order article via Infotrieve]
12. Li J, Culman J, Hortnagl H, Zhao Y, Gerova N, Timm M, Blume AK, E. Unger T, Zimmermann M, Seidel K, Dirnagl U, Unger T. Angiotensin AT2 receptor protects against cerebral ischemia-induced neuronal injury. FASEB J. 2005; 19: 617–619.
13. Lucius R, Gallinat S, Rosenstiel P, Unger T, Sievers J. The angiotensin II type 2 receptor promotes axonal regeneration in the optic nerve of adult rats. J Exp Med. 1998; 188: 661–670.
14. Laflamme L, Gasparo M, Gallo J, Payet M, Gallo-Payet N. Angiotensin II induction of neurite outgrowth by AT2 receptors in NG108-15 cells. J Biol Chem. 1996; 271: 22729–22735.
15. Reinecke K, Lucius R, Reinecke A, Rickert U, Herdengen T, Unger T. Angiotensin II accelerates functional recovery in the rat sciatic nerve in vivo: role of the AT2 receptor and the transcription factor NF-kB. FASEB J. 2003; 17: 2094–2096.
16. Gallinat S, Yu M, Dorst A, Unger T, Herdegen T. Sciatic nerve transection evokes lasting up-regulation of angiotensin AT2 and AT1 receptor mRNA in adult dorsal root ganglia and sciatic nerves. Brain Res Mol Brain Res. 1998; 57: 111–122.[Medline] [Order article via Infotrieve]
17. Widdop R, Jones E, Hannan R, Gaspari T. Angiotensin AT2 receptors: cardiovascular hope or hype? Br J Pharmacol. 2003; 140: 809–824.[CrossRef][Medline] [Order article via Infotrieve]
18. Jones E, Black MJ, Widdop RE. Angiotensin AT2 receptor contributes to cardiovascular remodelling of aged rats during chronic AT1 receptor blockade. J Mol Cell Cardiol. 2004; 37: 1023–1030.[CrossRef][Medline] [Order article via Infotrieve]
19. Lu Q, Zhu Y-Z, Wong PT-H. Neuroprotective effects of candesartan against cerebral ischemia in spontaneously hypertensive rats. Neuroreport. 2005; 16: 1963–1967.[CrossRef][Medline] [Order article via Infotrieve]
20. Iwai M, Liu HW, Chen R, Ide A, Okamoto S, Hata R, Sakanaka M, Shiuchi T, Horiuchi M. Possible inhibition of focal cerebral ischemia by angiotensin II type 2 receptor stimulation. Circulation. 2004; 110: 843–848.
21. Mogi M, Li J-M, Iwanami J, Min L-J, Tsukuda K, Iwai M, Horiuchi M. Angiotensin II type-2 receptor stimulation prevents neural damage by transcriptional activation of methyl methanesulfonate sensitive 2. Hypertension. 2006; 48: 141–148.
22. Thone-Reineke C, Steckelings U, Unger T. Angiotensin receptor blockers and cerebral protection in stroke. J Hypertens. 2006; 24: S115–S121.
23. Sharkey J, Ritchie I, Kelly P. Perivascular microapplication of endothelin-1: a new model of focal cerebral ischaemia in the rat. J Cereb Blood Flow Metab. 1993; 13: 865–871.[Medline] [Order article via Infotrieve]
24. Callaway JK, Knight MJ, Watkins DJ, Beart PM, Jarrott B. Delayed treatment with AM-36, a novel neuroprotective agent, reduces neuronal damage after endothelin-1 induced middle cerebral artery occlusion in conscious rats. Stroke. 1999; 30: 2704–2712.
25. Roulston CL, Callaway JK, Jarrott B, Woodman OL, Dusting GJ. Using behaviour to predict stroke severity in conscious rats: post-stroke treatment with 3',4'-dihydroxyflavonol improves recovery. Eur J Pharmacol. 2008; 584: 100–110.[CrossRef][Medline] [Order article via Infotrieve]
26. Metz GAD, V. Schwab ME, van de Meent H. The effect of unilateral pyramidal tract section on hindlimb motor performance in rats. Behav Brain Res. 1998; 96: 37–46.[CrossRef][Medline] [Order article via Infotrieve]
27. Callaway JK, Knight MJ, Watkins DJ, Beart PM, Jarrott B, Delaney PM. A novel, rapid, computerised method for quantification of neuronal damage in a rat model of stroke. J Neurosci Methods. 2000; 102: 53–60.[CrossRef][Medline] [Order article via Infotrieve]
28. Osborne KA, Shigeno T, Balarsky AM, Ford I, McCulloch J, Teasdale GM, Graham DI. Quantitative assessment of early brain damage in a rat model of focal cerebral ischaemia. J Neurol Neurosurg Psychiatry. 1987; 50: 402–410.
29. Touyz RM. Reactive oxygen species and angiotensin II signaling in vascular cells- implications in cardiovascular disease. Braz J Med Biol Res. 2004; 37: 1263–1273.[Medline] [Order article via Infotrieve]
30. Roulston CL, Jarrott AB, Widdop R. Localization of AT2 receptors in the nucleus of the solitary tract of spontaneously hypertensive and Wistar Kyoto rats using [125I]CGP42112: upregulation of a non-angiotensin II binding site following unilateral nodose ganglionectomy. Brain Res. 2003; 968: 139–155.[CrossRef][Medline] [Order article via Infotrieve]
31. Fisher M, Garcia J. Evolving stroke and the ischemic penumbra. Neurology. 1996; 47: 884–888.
32. Barber MN, Sampey DB, Widdop RE. AT2 receptor stimulation enhances antihypertensive effects of at1 receptor antagonist in hypertensive rats. Hypertension. 1999; 34: 1112–1116.
33. Li XC, Widdop RE. AT2 receptor-mediated vasodilatation is unmasked by AT1 receptor blockade in conscious SHR. Br J Pharmacol. 2004; 142: 821–830.[CrossRef][Medline] [Order article via Infotrieve]
34. Roulston CL, Lawrence AJ, Jarrott B, Widdop RE. Non-angiotensin II [125I] CGP42112 binding is a sensitive marker of neuronal injury in brainstem following unilateral nodose ganglionectomy: comparison with markers for activated microglia. Neuroscience. 2004; 127: 753–767.[CrossRef][Medline] [Order article via Infotrieve]
35. de Oliveira AM, Viswanathan M, Heemskerk FM, Correa FM, Saavedra JM. Specific, non-angiotensin, [125I]CGP 42112 binding sites in rat spleen macrophages. Biochem Biophys Res Commun. 1992; 200: 1049–1058.[CrossRef]
36. Zhu YZ, Chimon GN, Zhu YC, Lu Q, Li B, Hu HZ, Yap EH, Lee HS, Wong PT. Expression of angiotensin II AT2 receptor in the acute phase of stroke in rats. Neuroreport. 2000; 11: 1191–1194.[Medline] [Order article via Infotrieve]
37. Kreisel SH, Bazner H, Hennerici MG. Pathophysiology of stroke rehabilitation: temporal aspects of neurofunctional recovery. Cerebrovasc Dis. 2006; 21: 6–17.[CrossRef][Medline] [Order article via Infotrieve]
38. Miller AA, Dusting GJ, Roulston CL, Sobey CG. NADPH-oxidase activity is elevated in penumbral and non-ischemic cerebral arteries following stroke. Brain Res. 2006; 1111: 111–116.[CrossRef][Medline] [Order article via Infotrieve]
39. Hamai M, Iwai M, Ide A, Tomochika H, Tomono Y, Mogi M, Horiuchi M. Comparison of inhibitory action of candesartan and enalapril on brain ischemia through inhibition of oxidative stress. Neuropharmacology. 2006; 51: 822–828.[CrossRef][Medline] [Order article via Infotrieve]
Supplemental Materials and Methods
|
Surgical Procedures |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferencesSurgical ProceduresTreatmentsSystolic Blood PressureStroke InductionAssessment of Motor FunctionQuantification of Ischemic...Neuronal ImmunohistochemistryLocalization of Superoxide by...Angiotensin 2 Receptor...Statistical Analysis |
|---|
Male 15- to 16-week-old (270 to 320 g) SHR (Animal Resources Centre, Perth, Western Australia) were anesthetized with a ketamine (75 mg/kg; Sigma)/xylazine (10 mg/kg; Troy Laboratories) with additional doses administered as required. A 23-gauge stainless steel guide cannula was stereotaxically implanted into the piriform cortex 2 mm dorsal to the right middle cerebral artery according to a procedure previously described.23,24 The stereotaxic coordinates were modified for this strain of rat (0.2 mm anterior, –4.7 mm lateral, and –7 mm ventral relative to bregma). An additional 23-gauge ICV cannula was also stereotaxically implanted into the left lateral ventricle (–0.8 mm anterior, +1.5 mm lateral, and –3.2 mm ventral relative to bregma). The ICV cannula was attached to a drug-filled osmotic minipump (Alzet model 2ML2) through polyvinyl chloride tubing (0.50 mm inside diameter; Dural Plastics) and was buried subcutaneously. The guide cannulae were secured to the skull with dental acrylate cement and the incision closed with sutures. The animals were housed individually and were allowed a 5-day recovery period before the induction of stroke. These studies were approved by a Monash University Animal Ethics Committee.
|
Treatments |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferencesSurgical ProceduresTreatmentsSystolic Blood PressureStroke InductionAssessment of Motor FunctionQuantification of Ischemic...Neuronal ImmunohistochemistryLocalization of Superoxide by...Angiotensin 2 Receptor...Statistical Analysis |
|---|
Rats were randomly allocated to the following treatment groups: 0.1 (n=6), 1 (n=9), or 10 ng/kg/min CGP42112 (n=6; gift from Novatis); CGP42112 (1 ng/kg/min)+PD123319 (36 ng/kg/min; n=10; gift from Pfizer); PD123319 (36 ng/kg/min) alone (n=10); or saline (n=11). The ICV cannula and osmotic minipump containing these treatments were implanted 5 days before stroke and continued to deliver treatment for 3 days poststroke. Two additional sham groups, with or without CGP 42112 1 ng/kg/min, underwent surgery without stroke induction. These sham groups acted as surgical and histological controls, respectively.
|
Systolic Blood Pressure |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferencesSurgical ProceduresTreatmentsSystolic Blood PressureStroke InductionAssessment of Motor FunctionQuantification of Ischemic...Neuronal ImmunohistochemistryLocalization of Superoxide by...Angiotensin 2 Receptor...Statistical Analysis |
|---|
SBP was measured using a noninvasive tail-cuff apparatus (AD Instruments). At least 3 measurements were averaged on the day before the start of ICV treatment and also 72 hours poststroke (Day 14).
|
Stroke Induction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferencesSurgical ProceduresTreatmentsSystolic Blood PressureStroke InductionAssessment of Motor FunctionQuantification of Ischemic...Neuronal ImmunohistochemistryLocalization of Superoxide by...Angiotensin 2 Receptor...Statistical Analysis |
|---|
Stroke was induced in conscious animals by titrating the administration of the vasoconstrictor, ET-1 (Auspep), to cause temporary occlusion of the right middle cerebral artery.23,24 ET-1 was infused through a 30-gauge injector protruding 3 mm beyond the end of the previously implanted guide cannula. ET-1 (20 pmol/µL in saline) was administered at a rate of 0.2 µL every 30 seconds until the animal exhibited typical stroke behavior. Approximately 4 µL of ET-1 was infused to each animal with no significant difference in the dose of ET-1 administered between treatment groups. Stroke was characterized by behavioral indicators24,25 modified to be specific for the SHR strain. Typical behaviors that were observed were continuous contralateral or ipsilateral circling behavior; clenching, dragging, or failure to extend the forelimb contralateral to the side of ET-1 infusion; and forepaw shuffling, jaw flexing, or chin rubbing while circling. Each stroke was graded based on these predetermined behavioral changes using a scale of 1 to 4 with 1 being a mild stroke and 4 being a severe stroke. Core body temperature was monitored rectally using a digital thermometer over a 3-hour period. The experimenter was blinded to the treatments. Only rats with a stroke grade of 4, exhibiting at least 7 of the previously mentioned behaviors, were used for the purpose of this investigation, and any animal with a core temperature >40°C after stroke was euthanized.
|
Assessment of Motor Function |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferencesSurgical ProceduresTreatmentsSystolic Blood PressureStroke InductionAssessment of Motor FunctionQuantification of Ischemic...Neuronal ImmunohistochemistryLocalization of Superoxide by...Angiotensin 2 Receptor...Statistical Analysis |
|---|
Motor coordination and limb function were assessed using the ledged beam as previously described.26 The beam was constructed from wood and consisted of a 1.5-m long central section, which tapered from 9 cm to 1.5 cm from one end to the other. Underhanging ledges 1.5 cm in width were positioned 1.0 cm below the upper surface of the beam. The beam was placed at a 30° angle of incline with the highest point being the narrowest end, which led directly into the animal’s home cage. Rats were required to traverse the beam from the widest end to the narrowest, most difficult end. Animals were trained to traverse the beam on the day before the pretreatment assessment. The ledged beam test was conducted before treatment (Day 0), after recovery from cannula implantation (Day 6), immediately before stroke induction (Day 11), 24 hours poststroke induction (Day 12), and 72 hours poststroke induction (Day 14). All values were compared with presurgery; therefore, each rat acted as its own control. The number of total foot steps taken to run the length of the beam was recorded as well as the number of steps taken on the ledge by each foot. The latter number was recorded as a percentage of the total number of footsteps taken and recorded as percentage error.
|
Quantification of Ischemic Damage |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferencesSurgical ProceduresTreatmentsSystolic Blood PressureStroke InductionAssessment of Motor FunctionQuantification of Ischemic...Neuronal ImmunohistochemistryLocalization of Superoxide by...Angiotensin 2 Receptor...Statistical Analysis |
|---|
At 72 hours after stroke rats, were reanesthetized and perfused (25 mL/min) with physiologically buffered saline (0.1 mol/L; pH 7.4) through the apex of the heart. Brains were removed, frozen over liquid nitrogen, and stored at –80°C. Coronal cryostat sections (16 µm) were cut at 8 predetermined levels of the forebrain (–3.20 mm to 6.80 mm relative to bregma) slide mounted and stored at –80°C. The infarct area and volume were calculated using a method developed by Callaway and colleagues.27 The area of damage was measured by tracing around the area of infarct in each section using a computerized image analysis system (MCID M4 image analyzer; Imaging Research Inc). The infarct volume was quantified by integrating the area of damage with the distance between each level.28
|
Neuronal Immunohistochemistry |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferencesSurgical ProceduresTreatmentsSystolic Blood PressureStroke InductionAssessment of Motor FunctionQuantification of Ischemic...Neuronal ImmunohistochemistryLocalization of Superoxide by...Angiotensin 2 Receptor...Statistical Analysis |
|---|
Neuron integrity was assessed using the neuronal marker NeuN. Frozen coronal cryostat sections (16 µm) were postfixed in paraformaldehyde (4%). Slides were washed in Tris-buffer (Sigma, pH 7.4) for 30 minutes and then incubated in a humidified chamber in 10% goat serum, 0.5% BSA, and 0.3% Triton X-100 in Tris buffer for 1 hour. Sections were then washed in Tris buffer and incubated overnight in a 1/500 dilution of mouse monoclonal anti-NeuN IgG antibody (Chemicon). Adjacent sections were incubated with the antibody diluting solution as a negative control. The next day, sections were washed in Tris buffer and incubated for 2.5 hours with the fluorescence labeled secondary antibody, Alexa 488 (Invitrogen). After the incubation period, sections were washed once again in Tris buffer, dried, and cover-slipped using antifade mounting reagent (Vector shield) to prevent signal deterioration. The sections were then imaged using a fluorescence imaging microscope. The number of NeuN-positive cells was counted within a 1-mm2 site in the infarcted and noninfarcted region of the ipsilateral hemisphere and 2 matched regions of the contralateral hemisphere using analySIS software (Soft Imaging System).
|
Localization of Superoxide by Dihydroethidium Staining |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferencesSurgical ProceduresTreatmentsSystolic Blood PressureStroke InductionAssessment of Motor FunctionQuantification of Ischemic...Neuronal ImmunohistochemistryLocalization of Superoxide by...Angiotensin 2 Receptor...Statistical Analysis |
|---|
Dihydroethidium (2 µmol/L; Invitrogen) was used as previously described29 to detect superoxide in frozen coronal cryostat sections (16 µm; –0.8 mm relative to bregma in vehicle). The intensity of fluorescence was quantified within a 1-mm2 site in the infarcted and noninfarcted region of the ipsilateral hemisphere and 2 matched regions in the contralateral hemisphere using analySIS software (Soft Imaging System).
|
Angiotensin 2 Receptor Immunohistochemistry |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferencesSurgical ProceduresTreatmentsSystolic Blood PressureStroke InductionAssessment of Motor FunctionQuantification of Ischemic...Neuronal ImmunohistochemistryLocalization of Superoxide by...Angiotensin 2 Receptor...Statistical Analysis |
|---|
AT2R expression was assessed as previously described by Roulston et al.30 Frozen coronal cryostat sections (16 µm) were acetone-fixed. Slides were washed in Tris buffer (Sigma, pH 7.4) before incubation in a humidified chamber in 10% goat serum in Tris buffer for 30 minutes. This was followed by 30-minute incubation with peroxidase blocker (DAKO) to block nonspecific binding. Sections were then incubated overnight in a 1/250 dilution of rabbit polyclonal anti-AT2 IgG antibody (Santa Cruz). Adjacent sections were incubated with a rabbit immunoglobulin fraction (DAKO), used at the same antibody protein concentration, as a negative control. The next day, sections were washed in Tris buffer x3 and incubated for 2 hours with a peroxidase-labeled polymer conjugated to goat anti-rabbit immunoglobulins. After the 2-hour incubation, slides were washed in Tris buffer and then incubated with diaminobenzidine for 5 minutes. Sections were rinsed in distilled water, counterstained with hematoxylin, and cover-slipped. Sections were then imaged using a light microscope at 20x magnification. Images were taken from the cortical noninfarcted and infarcted regions of the ipsilateral hemisphere and 2 matched regions of the contralateral hemisphere. The number of cells immunopositive for AT2R was counted within a 0.25-mm2 site for each region.
|
Statistical Analysis |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferencesSurgical ProceduresTreatmentsSystolic Blood PressureStroke InductionAssessment of Motor FunctionQuantification of Ischemic...Neuronal ImmunohistochemistryLocalization of Superoxide by...Angiotensin 2 Receptor...Statistical Analysis |
|---|
Results are presented as mean±SEM. Ledged beam and blood pressure data were analyzed using 2-way repeated measures ANOVA with individual differences assessed using Bonferroni posttest. Infarct area, AT2R expression, neuronal expression, and superoxide production were assessed using one-way ANOVA with individual differences assessed using Dunnett’s posttest. All statistical analyses were performed using PRISM (GraphPad), and a value of P<0.05 was considered statistically significant.
|
Acknowledgments |
|---|
C.A.M. is the recipient of an Australian Postgraduate Award and a Monash University Faculty of Medicine Research Excellence scholarship.
Disclosures
None.
This article has been cited by other articles:
 |
|
 |
|
  K. Tsuda Angiotensin AT2 Receptor-Mediated Neuroprotection and Nitric Oxide-Bioavailability in Stroke Stroke, July 1, 2009; 40(7): e493 - e493. [Full Text] [PDF]
|
|
|
|
|
|
C. A. McCarthy, A. Vinh, J. K. Callaway, and R. E. Widdop Response to Letter by Tsuda Stroke, July 1, 2009; 40(7): e494 - e494. [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||