The phosphodiesterase-4 inhibitor rolipram protects from ischemic stroke in mice by reducing blood–brain-barrier damage, inflammation and thrombosis

Highlights

• Rolipram reduces stroke severity in mice in a clinically relevant time window.

• The underlying mechanisms include reduced inflammation, apoptosis and thrombosis.

• We propose PDE inhibitors as promising compounds for the treatment of ischemic stroke.

Abstract

Blood–brain-barrier (BBB) disruption, inflammation and thrombosis are important steps in the pathophysiology of acute ischemic stroke but are still inaccessible to therapeutic interventions. Rolipram specifically inhibits the enzyme phosphodiesterase (PDE) 4 thereby preventing the inactivation of the intracellular second messenger cyclic adenosine monophosphate (cAMP). Rolipram has been shown to relief inflammation and BBB damage in a variety of neurological disorders. We investigated the therapeutic potential of rolipram in a model of brain ischemia/reperfusion injury in mice. Treatment with 10 mg/kg rolipram, but not 2 mg/kg rolipram, 2 h after 60 min of transient middle cerebral artery occlusion (tMCAO) reduced infarct volumes by 50% and significantly improved clinical scores on day 1 compared with vehicle-treated controls. Rolipram maintained BBB function upon stroke as indicated by preserved expression of the tight junction proteins occludin and claudin-5. Accordingly, the formation of vascular brain edema was strongly attenuated in mice receiving rolipram. Moreover, rolipram reduced the invasion of neutrophils as well as the expression of the proinflammatory cytokines IL-1β and TNFα but increased the levels of TGFβ-1. Finally, rolipram exerted antithrombotic effects upon stroke and fewer neurons in the rolipram group underwent apoptosis. Rolipram is a multifaceted antiinflammatory and antithrombotic compound that protects from ischemic neurodegeneration in clinically meaningful settings.

Introduction

Ischemic stroke shows a complex pathophysiology that involves a plethora of distinct molecular and cellular pathways. Only recently the importance of inflammatory mechanisms in stroke has been recognized (Iadecola and Anrather, 2011, Magnus et al., 2012). Activation of cerebral endothelial cells represents one of the earliest events within the detrimental cascade of an ischemic insult causing upregulation of specialized cell adhesion receptors. Subsequently, blood-born inflammatory cells (e.g. neutrophils, macrophages) adhere to these receptors and invade the brain parenchyma across the blood–brain-barrier (BBB) in a coordinated and timed fashion. Those cells together with resident brain cells (e.g. microglia, endothelial cells) then secrete a potpourri of highly active soluble mediators like cytokines and chemokines that amplify the inflammatory response by attracting further immune cells or by causing direct tissue damage and neuronal apoptosis (Albert-Weißenberger et al., 2013). If the ischemic trigger persists, the structural components forming the BBB such as tight-junction proteins become disintegrated causing BBB leakage and finally, formation of vascular edema (Ayata and Ropper, 2002, Weiss et al., 2009). Brain edema is an important problem in the care of stroke patients as it can harm otherwise healthy brain areas by compression thereby inducing secondary functional deterioration and mortality. Until now, effective pharmacological strategies to counteract BBB damage and successive inflammation and edema formation in acute ischemic stroke are not available (Bardutzky and Schwab, 2007).

Progressive thrombus formation in the cerebral microvasculature is another important mechanism that mediates secondary infarct growth (Kraft et al., 2012). We could recently show that blocking of fibrin formation or platelet activation reliably protects from ischemic neurodegeneration in rodents (Hagedorn et al., 2010, Kleinschnitz et al., 2006, Kleinschnitz et al., 2007). Most notably, there is accumulating evidence of a subtle interplay between thrombosis and inflammation during the course of an ischemic insult, and this “thrombo-inflammation” might be suitable as novel therapeutic target (Nieswandt et al., 2011).

Rolipram is a selective phosphodiesterase-4 (PDE4) inhibitor that increases intracellular cyclic adenosine monophosphate (cAMP) levels in many cell types and tissues including the brain (Dyke and Montana, 2002). Initially developed as an antidepressant, rolipram also exerts substantial antiinflammatory and barrier stabilizing effects in the central nervous system (CNS) (Zhu et al., 2001). Treatment of rodents with rolipram for example protected from experimental autoimmune encephalomyelitis (EAE) by reducing BBB disruption and tumor necrosis factor α (Tnfα) and interferon γ (Ifnγ) production from autoreactive T cells (Folcik et al., 1999, Sommer et al., 1995). Beneficial effects of rolipram have also been described after experimental traumatic brain injury or spinal cord injury in rodents (Atkins et al., 2007, Beaumont et al., 2009, Costa et al., 2013, Kajana et al., 2009, Nikulina et al., 2004, Pearse et al., 2004, Whitaker et al., 2008) although few reports postulated detrimental or neutral effects of rolipram under these disease conditions (Atkins et al., 2012, Atkins et al., 2013, Wang et al., 2006). In models of global cerebral ischemia, rolipram diminished the area of tissue damage and improved memory deficits by attenuating neuronal loss in the hippocampus (Block et al., 1997, Li et al., 2011).

We here evaluated the efficacy and modes of rolipram action after transient middle cerebral artery occlusion (tMCAO) in mice, a model of focal brain ischemia/reperfusion injury.