Minocycline protects the blood–brain barrier and reduces edema following intracerebral hemorrhage in the rat
Introduction
Intracerebral hemorrhage (ICH) accounts for 10–30% of all strokes, with as much as 30–40% mortality by one month (Rincon and Mayer, 2004). After ICH, cerebral edema begins within hours, can last for weeks and is thought to contribute to neurological deterioration by increasing intracranial pressure or causing a shift in midline structures (Mayer and Rincon, 2005). Vasogenic edema is prevalent after ICH (Rincon and Mayer, 2004) and results from pathological changes in the blood–brain barrier (BBB) and consequent fluid movement from the vasculature to the extracellular space of the brain. Less important is the cytotoxic edema resulting from increased water uptake by brain cells (especially neurons). The BBB serves as the anatomical barrier between the blood and brain parenchyma and is comprised of endothelial cells, astrocytic end-feet, pericytes and a specialized basement membrane (the basal lamina) that serves as a scaffold supporting the physical integrity of the microvasculature. Although the basal lamina is clearly involved in BBB breakdown after ischemic stroke (Hamann et al., 2002), its role after ICH remains unresolved.
The inflammatory response following ICH, like other forms of brain injury, is characterized by production of several molecules that can disrupt the BBB (Wang and Dore, 2006). Moreover, because the delayed onset of both inflammation and vasogenic edema allows more time for patients to reach hospital after the onset of ICH, they are attractive therapeutic targets. In other forms of brain injury, the initial inflammatory response is orchestrated by the cytokines, TNFα and IL1β, which are up-regulated within hours (Barone and Feuerstein, 1999, Hua et al., 2006, Mayne et al., 2001b, Mayne et al., 2001a, Qureshi et al., 2001), but less is known about their temporal and spatial expression after ICH. While the ‘resting’ BBB severely limits movement of fluid, macromolecules and cells into the brain, TNFα and IL1β ‘activate’ the endothelium, increase its permeability and allow adhesion and entry of peripheral immune cells (Feuerstein et al., 1998, Middleton et al., 2002, Zhang et al., 2000). Transmigrating neutrophils in particular, produce cytotoxic molecules including inflammatory cytokines, reactive oxygen species and matrix metalloproteinases (MMPs) (Scholz et al., 2007). MMP production, also ascribed to endothelial cells, microglia/macrophages, astrocytes and neurons, can further damage the BBB by degrading the basal lamina of cerebral blood vessels (Rosenberg, 2002). Following ICH in the rat, several MMPs are up-regulated (Power et al., 2003, Wells et al., 2005), but their impact on cerebral blood vessels and BBB breakdown has yet to be assessed.
Minocycline, a semi-synthetic tetracycline derivative, is of particular therapeutic interest for CNS disorders because it has a high oral bioavailability, superior BBB penetration and is well tolerated by humans, where it has been used for decades to treat bacterial infections (Stirling et al., 2005, Yong et al., 2004). Its efficacy has been demonstrated in animal models of acute brain injury, including focal ischemia (Yrjanheikki et al., 1998, Yrjanheikki et al., 1999), ICH (Power et al., 2003), traumatic brain injury (Sanchez Mejia et al., 2001) and spinal cord injury (Wells et al., 2003), and proposed mechanisms include very diverse effects on several cell types. With respect to inflammation, it can inhibit activation and migration of inflammatory cells, and after ICH in the rat it inhibits macrophages/microglia (Power et al., 2003, Wasserman and Schlichter, 2007) and neutrophils (Wasserman and Schlichter, 2007). Minocycline has been reported to reduce specific cytokines (TNFα, IL1β) and MMPs (Elewa et al., 2006, Power et al., 2003) that have been implicated in BBB damage, and in animal models of ischemic stroke it can reduce BBB damage (Maier et al., 2006, Yenari et al., 2006).
The present work builds on two studies of collagenase-induced ICH in the rat striatum. Power and colleagues (2003) described the time course of MMP mRNA expression distal to the hematoma (2–4 mm) and effects of early treatment with minocycline (starting at 1 h) on MMP up-regulation and the neurological outcome. In the same model (Wasserman and Schlichter, 2007), we recently investigated the spatial and temporal relationship between neuron death and inflammation, and effects of minocycline treatment begun at a later, more clinically relevant time (6 h); i.e., after the hematoma had reached its maximal size. Using the same model, we now assess the effects of delayed minocycline treatment on blood vessel deterioration and edema, and on time-dependent changes in several key molecules involved in these events. BBB damage was monitored by counting the number of intact microvessels surrounding the hematoma (Hamann et al., 2002), assessing the extravasation of IgG into the brain (Tanno et al., 1992), and measuring brain water content 3 days after ICH onset. Then, because a prominent inflammatory response occurs in the tissue immediately adjacent to the hematoma, we monitored gene expression in and around the damage site with and without minocycline treatment and began at an earlier time than previously examined. Expression of IL1β, TNFα, MMP-3, MMP-9, MMP-12 and the microglia/macrophage marker, complement receptor 3 were monitored at 6 h, 1 day and 3 days after ICH, using quantitative real-time RT-PCR. Finally, for the two genes whose up-regulation was reduced by minocycline (TNFα, MMP-12), we used immunohistochemistry to determine which cell types expressed the proteins. We show that TNFα increased early, mainly in neutrophils; whereas, MMP-12 increased later in damaged microvessels surrounding the hematoma. Delayed minocycline treatment reduced: microvessel loss, plasma protein extravasation and edema; expression of TNFα and MMP-12; and the number of TNFα-positive cells and neutrophils in the brain. These findings support a model whereby immune cells produce TNFα, which leads to an increase in MMP-12 in microvessels surrounding the hematoma, causing further degradation of the basal lamina and BBB disruption.
Section snippets
Intracerebral hemorrhage and minocycline treatment
Intracerebral hemorrhage was induced in the striatum of male Sprague–Dawley rats (300–350 g, n = 40) as previously described (Rosenberg et al., 1990, Wasserman and Schlichter, 2007). All procedures were approved by the University Health Network animal care committee, in accordance with guidelines established by the Canadian Council on Animal Care. Rats were anesthetized using isoflurane (3% induction, 1.5% maintenance) and placed in a stereotaxic frame. Under aseptic conditions, a 1-mm diameter
Minocycline reduces cerebral edema and the loss of microvessels
Cerebral edema peaks at 3–4 days after ICH onset in the rat (see Discussion); thus, we measured the brain water content at 3 days (Fig. 1B). Naïve rats, not subjected to any surgical procedure, had a brain water content of 78.65 ± 0.11% (n = 6), whereas sham-operated rats (saline-injected, no collagenase; n = 3) had a brain water content of 79.2 ± 0.09% in the ipsilateral hemisphere and 78.9 ± 0.06 in the contralateral hemisphere. The cerebral hemisphere was used as the internal control, rather than the
Discussion
This work presents several novel findings. The results show for the first time that minocycline treatment, delayed to a clinically relevant starting time, protects the blood–brain barrier (BBB) after ICH. This protection was manifested by amelioration of three key processes; i.e., decreases in the number of disrupted microvessels, serum protein extravasation into the brain and cerebral edema. This is apparently the first report using collagen type IV expression in order to show reduced numbers
Acknowledgments
Excellent technical help was provided by X-P Zhu and H Yang. We are grateful for comments from Drs. J Eubanks, L Mills and EF Stanley. Supported by grants to LCS from the Heart and Stroke Foundation (HSFO; NA5158, T5546), Krembil Scientific Development Seed Fund, Canadian Stroke Network and a scholarship to JW from HSF Canada.
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