Traumatic brain injury is characterized by pathophy siological processes such as excitotoxicity, oxidative stress, inflammation, neuro-immunity, and neurodegen eration in the early phase (Cornelius et al., 2013; 22 Algattas and Huang, 2014; Kelso and Gendelman, 23 2014), and it is characterized by neuronal regeneration and the activation of astrocytes and microglia in the chronic phase (Algattas and Huang, 2014). Although 26 extensive efforts have been made to explore the mechanisms underlying traumatic brain injury and to identify therapeutic targets (Jennings et al., 2008), the effects of pharmacological intervention on traumatic brain injury are still limited.
Nicotinamide phosphoribosyltransferase (NAMPT, also known as pre-B cell enhancing factor or visfatin) is the key enzyme in the salvaging pathway for the biosynthesis of nicotinamide adenine dinucleotide (NAD). NAMPT catalyzes the synthesis of nicotinamide mononucleotide (NMN), which is further converted to NAD (Rongvaux et al., 2002). NAD is a co-factor for NAD-dependent enzymes such as sirtuin 1 and poly [ADP-ribose] polymerase 1 (Rajamohan et al., 2009), and is involved in energy metabolism (Wilson, 1994; Hertz, 2008). Thus, NAD participates in many cellular functions (Zhang and Kraus, 2010), and modulating the level of NAD is an attractive target for the treatment of many diseases (Khan et al., 2007; Magni et al., 2009).
In the brain, NAMPT is mainly expressed in neurons. It has been shown that NAMPT protects the brain from ischemic injury via the synthesis of NAD. Decreasing the level of NAD by knocking down the expression of NAMPT or using NAMPT inhibitors aggravates ischemic injury in mice (Zhang et al., 2010) and rats (Wang et al., 2011), whereas increasing the level of NAD by overexpression of NAMPT or using NMN reduces ischemiainduced brain injury in rats (Wang et al., 2011) and in primary cultured neurons (Bi et al., 2012).
On the other hand, NAMPT is also widely expressed in inflammatory cells, and is responsible for the activation of monocytes (Schilling et al., 2012), neutrophils (Montecucco et al., 2013a; Nencioni et al., 2014) and macrophages (Venter et al., 2014) via the synthesis of NAD. NAMPT has been identified as an inflammatory factor (Sun et al., 2013; Montecucco et al., 2013b,c), and it has been shown that inhibiting the biosynthesis of NAD in inflammatory cells with NAMPT inhibitors has therapeutic effects in inflammation-related diseases including arthritis (Busso et al., 2008), myocardial infarction (Montecucco et al., 2013a), and atherosclerosis (Nencioni et al., 2014). Inflammation is one of the most important pathophysiological processes after traumatic brain injury, and can cause brain damage (Woodcock and MorgantiKossmann, 2013). The cellular inflammation is characterized by the infiltration of peripheral inflammatory cells like neutrophils and macrophages in the early phase, and by the activation of macrophages/microglia and astrocytes in both the early and chronic phases (Soares et al., 1995; Jin et al., 2012). Since NAD is involved both in the activation of inflammatory cells and in the protection of neurons from ischemic injury, we set out to understand the effects of increasing and decreasing the level of NAD on traumatic brain injury. Cryoinjury is an established animal model that mimics traumatic brain injury in some pathophysiological processes (Covey et al., 2010; Kim et al., 2013). Here we used FK866 (a potent NAMPT inhibitor) to reduce the NAD level, and used Bioavailable NMN (the product of NAMPT and precursor of NAD) to increase the NAD level, and we determined their effects on brain injury in the mouse cryoinjury model.
A total of 116 male Balb/c mice (25–30 g, 8–9 weeks old) were purchased from the Experimental Animal Center of Zhejiang Academy of Medicine Sciences. All procedures were carried out in accordance with the Guide for the Care and Use of the Laboratory Animals of the National Institutes of Health. The experimental protocols were approved by the Ethics Committee of Laboratory Animal Care and Welfare, School of Medicine, Zhejiang University. All animals were anesthetized by intrape ritoneal injection of 10% chloral hydrate before sacrifice, and efforts were made to minimize suffering. Intracerebroventricular administration Forty-eight hours before cryoinjury, mice were anesthetized by intraperitoneal injection of chloral hydrate (400 mg/kg). The mice were then fixed on a stereotactic frame (SR-5, Narishige, Tokyo, Japan). A hole (1 mm lateral to midline and 0.5 mm from bregma) was carefully drilled in the skull. Then either 7-ll saline, or 5 lM FK866 in 7-ll saline, or 5 mM NMN in 7-ll saline was injected into the lateral ventricle (3.0 mm below dura) in 10min using a micro-injector. Control mice were anesthetized and a hole was drilled in the skull, but there was no injection. The dose of NMN was chose according to the literature, in which 30mM NMN in 2-ll saline was injected into the lateral ventricle of rats (Wang et al., 2011). For FK866, our preliminary data showed that 1 nM to 10 lM FK866 in 7-ll saline decreased the NAD level in the mouse brain cortex in a dose-dependent manner (data not shown). However, the injection of 10 lM but not 5 lM FK866 resulted in neuronal injury (data not shown).