Elsevier

Brain Research

Volume 1208, 7 May 2008, Pages 225-233
Brain Research

Research Report
Comparison between single and combined treatment with candesartan and pioglitazone following transient focal ischemia in rat brain

https://doi.org/10.1016/j.brainres.2008.02.032Get rights and content

Abstract

Angiotensin AT1 receptor blockers (ARBs) and thiazolidinediones (TZDs) have become well established drugs for the treatment of major risk factors of stroke. Since several studies provided evidence that ARBs and TZDs also have additional anti-inflammatory effects, we hypothesized that a combined treatment with the ARB, candesartan, and the TZD, pioglitazone, ameliorates ischemia-induced brain injury and inflammation by synergistic anti-inflammatory actions. Normotensive Wistar rats were pre-treated for 5 days with vehicle (0.9% NaCl), 0.2 mg/kg/day candesartan (s.c.), and/or 2 and/or 20 mg/kg/day pioglitazone (p.o.), respectively and underwent 90 min of middle cerebral artery occlusion (MCAO) with successive reperfusion. Neurological deficits and infarct size were determined 24 h and 48 h after MCAO, respectively, followed by tissue sampling. Animals treated with candesartan, pioglitazone, and the combination of candesartan and pioglitazone had reduced neurological deficits 24 h and 48 h after MCAO, respectively (P < 0.05–0.01). Infarct size was reduced by treatment of candesartan, pioglitazone, and their respective combination (each P < 0.05) 48 h after stroke compared to vehicle. Treatment with candesartan, pioglitazone, and their combination resulted in significantly reduced mRNA expression of the inflammatory markers CXCL1 and TNFα in vivo (P < 0.01). The combination of candesartan plus pioglitazone is equally effective compared to their single applications concerning neuroprotection and attenuation of inflammation after MCAO. Therefore, we conclude that a direct synergistic neuroprotective action of parallel ARB and TZD treatment is unlikely.

Introduction

Despite an improvement of clinical care in patients with cerebrovascular insult, stroke morbidity and mortality is still high (Murray and Lopez, 1996) and new neuroprotective pharmacological strategies are required. Hypertension and type 2 diabetes are key symptoms of the metabolic syndrome and two of the most important risk factors in developing cardiovascular disease, which subsequently can lead to stroke. Once a patient is suffering from one or more cardiovascular risk factors the major goals are to treat them and simultaneously to care for the underlying causes of the syndrome.

Several studies have provided evidence that angiotensin AT1 receptor blockers (ARBs) and thiazolidinediones (TZDs) have additional actions beyond their effects in the treatment of major stroke risk factors such as hypertension or type 2 diabetes (De Gasparo et al., 2000, Luo et al., 2006). In recent years, several clinical studies including LIFE, MOSES, SCOPE and ACCESS, have shown the benefit of ARBs in primary and secondary prevention of stroke (Schrader, 2005, Schrader, 2003, Lithell, 2002, Dahlöf et al., 2002). In experimental studies, neuroprotective effects of ARBs in cerebral ischemia were not only observed in hypertensive but also in normotensive animals and even with sub-antihypertensive doses. For instance, chronic systemic pre-treatment with candesartan reduced cerebral infarct size in both spontaneously hypertensive rats (SHR) and normotensive rats subjected to middle cerebral artery occlusion (MCAO) (Groth et al., 2003, Ito et al., 2002, Nishimura et al., 2000) indicating that ARBs can improve recovery from stroke by blood pressure-independent actions.

Peroxisome proliferator-activated receptors (PPARs) are nuclear receptors that function as ligand-activated transcriptional regulators of genes controlling lipid and glucose metabolism (Vamecq and Latruffe, 1999). TZDs are synthetic agonists of PPARγ that have been shown to reduce cerebral infarction volume and improve neurological function against transient focal ischemia (Zhao et al., 2005, Sundararajan et al., 2005).

Therefore, a combination therapy composed of the angiotensin AT1 receptor blocker (ARB), candesartan, and the thiazolidinediones (TZDs), pioglitazone, probably could be a successful therapeutic option for patients with metabolic syndrome in daily clinical care.

Cerebral ischemia is accompanied by an intense inflammatory response which is characterized by the expression of a broad range of pro-inflammatory mediators previously demonstrated to worsen ischemic brain injury, increase infarction size and impair neurologic outcome (Iadecola and Alexander, 2001). Angiotensin II (Ang II), the principal effector peptide of the renin–angiotensin system (RAS), was recently, in addition to its effects on blood pressure, also characterized as a pro-inflammatory mediator (Ferrario and Strawn, 2006), and experimental studies suggest that ARBs may have secondary anti-inflammatory properties (Dandona et al., 2003). Similarly, in addition to their antidiabetic effects, TZDs may protect against ischemic brain damage through an inhibitory action on inflammation (Daynes and Jones, 2002). Thus, several studies demonstrated that PPARγ agonists reduced CNS inflammation including macrophage/microglial activation following cerebral occlusion (Zhao et al., 2005, Sundararajan et al., 2005). These results suggest actions of TZDs modulating the pathophysiological events following stroke by suppressing the inflammatory response.

There is in vivo and in vitro evidence for a direct interaction between the RAS and PPARγ (Tham et al., 2002, Sugawara et al., 2001, Goetze et al., 1999). Furthermore, as shown in in vitro experiments combination of an ARB and a PPARγ agonist may have synergistic actions on different molecular functions. Activation of PPARγ by TZDs has been demonstrated to downregulate AT1 receptor expression (Sugawara et al., 2001) and, vice versa, AT1 receptor activation by Ang II to decrease vascular PPARγ (Tham et al., 2002), whereby AT1 receptor blockade would be expected to maintain PPARγ levels. Given the strong support for ARB/TZD synergism in vitro, the knowledge of in vivo interventions, in particular in a model of cerebral ischemia, is still limited. The question as to whether there are any relevant beneficial synergies between ARBs and TZDs with regard to their anti-inflammatory actions in brain ischemia has not yet been addressed.

Therefore, we analyzed the effects of a combined pre-treatment with an ARB, candesartan, and a PPARγ agonist, pioglitazone, on infarct size, neurological function and expression of inflammatory mediators in the brain of rats undergoing middle cerebral artery occlusion (MCAO). In agreement with previous studies, we report here that candesartan and pioglitazone, when applied individually, provided a potent protective effect against cerebral injury, and inhibited inflammatory response in the brain. Both, the ARB and the TZD, significantly reduced infarction size following MCAO and this reduction was accompanied by improved neurologic function. However, synergistic actions of a combination therapy with candesartan and pioglitazone were not observed, and, in most aspects, the combination was not different from the individual effects of both drugs.

Section snippets

Blood parameters

Oxygen, carbon dioxide, pH, glucose, haematocrit, sodium and potassium levels showed no significant differences at any time point between all 6 treatment groups (data not shown).

Infarct volume

Cerebral ischemic areas were visualized by MRI technique and their volume was determined. On day two after MCAO, the mean size of ischemic lesions was 401.6 ± 29.2 mm3 in vehicle-treated animals (Fig. 1). In rats pre-treated with the combination of candesartan and pioglitazone low dose, the mean size of the ischemic area

Discussion

In recent years, several studies have shown that systemic treatment with ARBs (Groth et al., 2003, Dai et al., 1999) or PPARγ ligands exerts various beneficial effects in cerebral ischemia (Zhao et al., 2005, Sundararajan et al., 2005). A potent method of mitigating the intense derangements associated with stroke could be a dual ARB/PPARγ interacting mechanism that is capable of integrating and modulating two major pathways, one through activation of PPARγ pathway, and the second by selectively

Animals

Male normotensive Wistar rats weighing 180–200 g were obtained from HARLAN Winkelmann (Borchen, Germany). Animals were kept in a SPF (specific pathogen free) barrier with respect to temperature and humidity, and were housed on a 12 h light/12 h dark cycle in groups of 4–5 with free access to food and water. Animal housing, care and experimental procedures complied with the Guide for the Care and Use of Laboratory Animals of the State Government of Berlin, Germany.

Drug administration

Treatment groups of animals

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