Cilostazol reduces blood brain barrier dysfunction, white matter lesion formation and motor deficits following chronic cerebral hypoperfusion

Highlights

• BBB permeability is significantly elevated for 2 weeks following permanent BCCAO in the rat.

• BCCAO was found to impair gait in the tapered beam test.

• Cilostazol, a type III phosphodiesterase inhibitor, reduced Evans blue dye extravasation and gait disturbance in vivo.

• Cilostazol reduces white matter rarefaction and gliosis in the optic tract.

• Cilostazol reduced plasma toxicity in vitro.

Abstract

Cerebral small vessel disease (CSVD) is a pathological process leading to lacunar infarcts, leukoaraiosis and cerebral microbleeds. Dysfunction of the blood brain barrier (BBB) has been proposed as a mechanism in the progression cerebral small vessel disease. A rodent model commonly used to study some aspects of CSVD is bilateral common carotid artery occlusion (BCCAO) in the rat. In the present study it was determined that gait impairment, as determined by a tapered beam test, and BBB permeability increased following BCCAO. Cilostazol, a type III phosphodiesterase inhibitor, has been shown to have anti-apoptotic effects and prevent white matter vacuolation and rarefaction induced by BCCAO in rats. In this study the protective effect of cilostazol administration on the increase BBB permeability following BCCAO was determined as well as the effect on plasma levels of circulating microparticles (MPs), cerebral white matter rarefaction, glial activation and gait disturbance. The effect of cilostazol on in vitro endothelial barriers was also evaluated. Cilostazol treatment improved BBB permeability and reduced gait disturbance, visual impairment and microglial activation in optic tract following BCCAO in vivo. It also reduced the degree of cell death and the reduction in trans-endothelial electrical resistance (TEER) in artificial endothelial barriers in vitro induced by MP treatment of in vitro barriers.

Introduction

Cerebral small vessel disease (CSVD) is now thought to be the most common cause of vascular cognitive impairment (VCI) and accounts for more than 40% of all VCI (Bowler, 2005). CSVD is associated with chronic cerebral hypoperfusion and the reduction in blood flow in the perforating arteries resulting in ischemia in the white matter and deep gray matter nuclei. The microangiopathy induced by CSVD is characterized radiologically by the presence of lacunes (microinfarcts of the central gray matter) and leukoaraiosis in various regions of the brain (Schmidtke and Hüll, 2005). CSVD is a salient risk factor for stroke (Wardlaw et al., 2013). CSVD represents a serious health care challenge, as lacunar stroke may occur with an incidence of up to five-times that of large vessel stroke (Vermeer et al., 2007). CSVD may result in cognitive impairment, especially affecting executive function, and mild motor impairments such as gait disturbance (de Laat et al., 2010). Disruption of white matter tracts that connect motor regions is thought to be the mechanism underlying the gait disturbance noted in some patient with CSVD (Chui, 2007), and WM lesions in certain brain regions such as subcortical areas and periventricular frontal lobe are related to lower gait velocity, shorter stride length and broader stride width (Silbert et al., 2008).

The endothelium plays a crucial role in processes such as the autoregulation of blood flow, reactive hyperemia and formation and function of the blood-brain barrier. A failure of the arteriolar endothelium due to partial ischemia has been proposed as an early event in the pathogenesis of CSVD (Pantoni and Garcia, 1997). Endothelial injury also leads to BBB leakage, which has been considered as the starting point of CSVD (Schreiber et al., 2013). This derangement in the blood brain barrier may initiate some years before the first symptoms of CSVD and leads to structural, vascular and perivascular changes in brain small vessels such as edema, enlarged perivascular spaces and tissue damage (Wardlaw, 2005, Wardlaw, 2010).

MPs are small vesicles, 0.1–1.0 µm in size, that bud off from plasma membrane of various cell types in response to shear stress, physiological agonizts such as collagen (Morel et al., 2011), and pro-apoptotic stimuli (György et al., 2011). MPs may also be released spontaneously from cells under basal condition (Italiano et al., 2010). MPs transfer information from the cell of origin to target cells by cell-to-cell contact or through secretion of soluble mediators (Mause and Weber, 2010). It has been proposed that MPs play a role in pathogenesis of cerebral malaria and multiple sclerosis through induction of brain endothelial dysfunction and alterations in BBB permeability (Combes et al., 2006, Minagar and Alexander, 2003). Circulating MPs are markers of early stages of multiple sclerosis and increase endothelial permeability (Marcos-Ramiro et al., 2014).

BCCAO is one of most widely used models for the study of CVSD (Hainsworth and Markus, 2008, Hainsworth et al., 2012). BCCAO in rats induces a chronic reduction in cerebral blood flow in frontal cortex for 8 weeks to 3 months after surgery (Ohta et al., 1997, Ueno et al., 2002, Farkas et al., 2007). Lesions are mainly in white matter (WM), and appear 7 days after occlusion and are persistent (Wakita et al., 1995; Ueno et al., 2002; Farkas et al., 2004). They are preceded by temporary BBB opening in white matter areas with collagen deposition in vessel walls (Ueno et al., 2002).

Cilostazol, a type III phosphodiesterase inhibitor, has been reported to be neuroprotective against apoptotic white matter damage and cognitive impairment in rats that received BCCAO (Lee et al., 2006, Watanabe et al., 2006, Lee et al., 2007, Kwak et al., 2012). It has been recently acknowledged as one of potential pharmacologic interventions for treatment of CSVD (Bath and Wardlaw, 2015).

It has been previously shown that the induction of chronic cerebral hypoperfusion in rats causes an increase in the number of MPs circulating in the plasma, and these MPs are able to induce cell death when they are applied to rat brain microvascular endothelial cells (Schock et al., 2014). MPs isolated from plasma of rats which received BCCAO also increase rat brain microvascular endothelial cell (RBMVECs) permeability when they are applied to in vitro artificial barriers (Edrissi et al., 2016).

The purpose of this study was to assess the role of increased BBB permeability in the progression of small vessel disease using this rat model of chronic cerebral hypoperfusion. The specific objectives were to study the extent and time course of changes in BBB permeability and motor impairment and to determine the effects of cilostazol administration on these processes. Cilostazol treatment reduced the effect of chronic ischemia on the BBB, improved gait performance in the tapered beam test and reduced the severity of lesions in vulnerable white matter regions (optic tract and corpus callosum) following BCCAO. Plasma toxicity was measured at 2 weeks following BCCAO in order to determine the association between the numbers of circulating MPs and the pathological consequences of cerebral ischemia.