TNF-α knockout and minocycline treatment attenuates blood–brain barrier leakage in MPTP-treated mice

Abstract

Following intraparenchymal injection of the dopamine (DA) neurotoxin 6-hydroxydopamine, we previously demonstrated passage of fluoresceinisothiocyanate-labeled albumin (FITC-LA) from blood into the substantia nigra (SN) and striatum suggesting damage to the blood–brain barrier (BBB). The factors contributing to the BBB leakage could have included neuroinflammation, loss of DA neuron control of barrier function, or a combination of both. In order to determine which factor(s) was responsible, we assessed BBB integrity using the FITC-LA technique in wild-type (WT), tumor necrosis factor alpha (TNF-α) knockout (KO), and minocycline (an inhibitor of microglia activation) treated mice 72 h following treatment with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Compared with WT mice, TNF-α KO mice treated with MPTP showed reduced FITC-LA leakage, decreased numbers of activated microglia, and reduced proinflammatory cytokines (TNF-α and interleukin 1β) associated with significant MPTP-induced DA neuron loss. In contrast, minocycline treated animals did not exhibit significant MPTP-induced DA neuron loss although their FITC-LA leakage, numbers of activated microglia, and MPTP-induced cytokines were markedly attenuated. Since both TNF-α KO and minocycline treatment attenuated MPTP-induced BBB dysfunction, microglial activation, and cytokine increases, but had differential effects on DA neuron loss, it appears that neuroinflammation and not DA neuron loss was responsible for disrupting the blood–brain barrier integrity.

Introduction

Parkinson’s disease (PD) is marked by the progressive loss of dopamine (DA) neurons in the substantia nigra (SN) (Hastings and Zigmond, 1997). While the etiology of PD remains unclear, both genetic factors and environmental toxins have been proposed in its pathogenesis (Ladeby et al., 2005, McGeer and McGeer, 2004). Regardless of the underlying etiology, neuroinflammation [the some total of cellular changes (e.g., microglial activation) and secreted factors (e.g., proinflammatory cytokines or free radicals) that accompany an inflammatory response within the CNS] is thought to contribute to the loss of DA neurons seen in patients with PD (McGeer and McGeer, 2004, Ladeby et al., 2005). Neuroinflammation is also present in trauma, stroke, multiple sclerosis, epilepsy and bacterial meningitis (Mennicken et al., 1999, Phillis et al., 2006) in which damage to the blood–brain barrier (BBB) has been reported (Huber et al., 2001). More recent studies point to microvascular changes in the SN and alterations in several markers of normal BBB integrity in PD patients (Barcia et al., 2004, Faucheux et al., 1999, Kortekaas et al., 2005). Whether actual changes in permeability, functionality, or physical damage of the BBB occur in PD is currently unknown.

We recently demonstrated that the DA neurotoxin, 6-hydroxydopamine (6-OHDA), compromised BBB integrity producing apparent leakage of both fluoresceinisothiocyanate-labeled albumin (FITC-LA) and horseradish peroxidase into the SN and striatum. This leakage was accompanied by loss of DA neurons, activation of microglia, up-regulation of p-glycoprotein and β-integrin on the endothelial cells that comprise the BBB, and attenuation of a DA-mediated behavior by domperidone, a DA antagonist that normally does not cross the BBB (Carvey et al., 2005). Since neuroinflammation including microglia activation and increased levels of proinflammatory cytokines including tumor necrosis factor-alpha (TNF-α) are present in patients with PD and in animal models of the disease (Aschner, 1998, Hirsch et al., 2005, Nagatsu and Sawada, 2005) and both are well known to affect the integrity of the BBB (Ryu and McLarnon, 2006, Tsao et al., 2001, Yenari et al., 2006), it is quite possible that 6-OHDA-induced neuroinflammation was responsible for the breakdown in barrier integrity. However, neurons containing biogenic amines are found in close proximity to brain capillaries (Rennels and Nelson, 1975, DiCarlo et al., 1984, Kapadia and de Lanerolle, 1984), endothelial cells express both noradrenergic and serotoninergic transporters (Wakayama et al., 2002) and receptors (Wakayama et al., 2002, Kobayashi et al., 1985), and stimulation of the locus coeruleus increases BBB permeability (Raichle et al., 1975) suggesting that neurotransmitters may regulate BBB function as well. The BBB leakage we observed in rats treated with 6-OHDA could be the result of neuroinflammation, DA neuron loss, or a combination of both. Therefore, we designed a set of experiments to determine the relative contribution of each to the leakage of the BBB.