Elsevier

Brain Research

Volume 1125, Issue 1, 13 December 2006, Pages 185-193
Brain Research

Research Report
The effects of nicotinamide on apoptosis and blood–brain barrier breakdown following traumatic brain injury

https://doi.org/10.1016/j.brainres.2006.10.019Get rights and content

Abstract

Nicotinamide has been shown to protect against many of the pathophysiological factors associated with both ischemic and traumatic brain injuries. The present study evaluated the neuroprotective effect of nicotinamide on the breakdown of the blood–brain barrier (BBB) and apoptosis expression following traumatic brain injury (TBI). Animals were prepared with a unilateral cortical contusion injury (CCI). Fifteen minutes following injury the animals received either nicotinamide (500 mg/kg, ip) or 0.9% saline. The animals were perfused at 5, 24, and 72 h post-injury. BBB integrity was assessed by endogenous rat IgG immunoreactivity. Recent studies have shown that IgG immunoreactivity is a reliable measure of BBB integrity. The results indicated that IgG immunoreactivity was greatest at 5 h and declined at 24 h after injury. Nicotinamide significantly reduced IgG expression at every time point following injury. Apoptosis was examined using the TUNEL method. The results indicated that TUNEL immunoreactivity peaked at 24 h. TUNEL+ cells were classified morphologically as nonapoptotic (Type I) or apoptotic (Type II) to verify that the neuroprotective effects of nicotinamide occur by inhibiting apoptosis or necrosis. Administration of nicotinamide significantly reduced the expression of all TUNEL+ cells in the tissue surrounding the lesion cavity. Specifically there was a significant reduction in the number of Type I, Type II, and Total TUNEL+ cells in the nicotinamide-treated animals. In addition, nicotinamide reduced lesion cavity expansion 72 h following CCI. These findings suggest that nicotinamide reduces BBB breach and neuronal cell loss acutely following injury and that these reductions may account for the beneficial behavioral effects seen in previous studies.

Introduction

Traumatic brain injury (TBI) is a major public health issue. Conservative estimates for TBI in the United States put the incidence rate at 200 per 100,000, yielding 500,000 cases annually, with 20% being classified by the Glasgow Coma Scale (GCS) as severe or moderate injuries (Narayan et al., 2002). These disabilities include cognitive, sensory, motor, and emotional impairments. This major health issue is compounded by the lack of pharmacological treatments that are currently available for patients suffering head injuries, which is surprising because of the amount of effort invested into finding such treatments (Narayan et al., 2002, Statler et al., 2001).

Brain damage following TBI is the result of both a primary and secondary injury. The primary, or direct, injury involves the immediate mechanical disruption of brain tissue. This results in loss of tissue, edema, and the tearing and/or shearing of axons within the brain, including cells composing the blood–brain barrier (BBB) (Lenzlinger et al., 2001). The secondary, indirect or delayed, stage involves several mechanisms, including the initiation of an acute inflammatory response, infiltration of peripheral blood cells, activation of resident immunocompetent cells, and the release of many immune mediators such as interleukins and chemotactic factors (Lenzlinger et al., 2001). The acute inflammatory response also includes edema formation, swelling, and the breakdown of the BBB (Lenzlinger et al., 2001).

The breakdown of the BBB is most likely caused by changes in the capillary endothelial cells or their tight junctions. A recent study has shown that TBI causes microvascular basal lamina damage in cortical areas, indicating that the BBB is disrupted during and after TBI (Muellner et al., 2003). In another study, the BBB was shown to have been broken down 1 h post-injury, but not at 6 h (Hartl et al., 1997). It has also been shown that edema peaked at 24 h following injury and then declined. It was shown that the BBB opened in a biphasic manner following a cortical contusion injury (CCI); however, the second opening did not contribute to any further increases in edema formation (Baskaya et al., 1997). The first opening was found to peak around 4 to 6 h following TBI and the second opening peaked around 3 days following TBI. This biphasic opening of the BBB has been found to coincide with the primary and secondary injury cascades (Baskaya et al., 1997). Thus, one of the important factors to consider following TBI is the breakdown of the BBB, which can simply be detected using IgG immunoreactivity (Tanno et al., 1992).

Another important secondary injury component is cell death mediated by apoptosis and/or necrosis. There are many factors that contribute to apoptosis (Raghupathi et al., 2000); however, poly (ADP-ribose) polymerase (PARP), a nuclear enzyme that aids in DNA repair, has been implicated as a major contributing factor. The over activation of PARP is detrimental and causes depletion of cellular NAD and ATP supplies, resulting in energy depletion and eventually apoptosis (Liaudet et al., 2003). Recent research has shown that PARP inhibition can prevent neurological and neuropathological deficits after TBI and ischemia (LaPlaca et al., 2001, Liaudet et al., 2003). Thus, PARP inhibition may be an important mechanism of action for novel neuroprotective agents.

An interesting novel therapy that may have beneficial effects on the pathophysiology of TBI and recovery of function is nicotinamide, a potent PARP inhibitor. Several studies have demonstrated the neuroprotective ability of nicotinamide following experimental focal cerebral ischemia (see Maiese and Chong, 2003, for a review). Multiple doses of nicotinamide were shown to provide neuroprotection against focal cerebral ischemia in rats for up to 2 weeks post-injury (Maynard et al., 2001). In another study, nicotinamide was shown to reduce neuronal infarction in a dose-specific manner following permanent middle cerebral artery occlusion, where a dose of 500 mg/kg was determined to be the most effective (Ayoub et al., 1999). Utilizing the same injury model, nicotinamide was also shown to improve neurological outcome by improving sensory and motor behavior following transient focal cerebral ischemia (Mokudai et al., 2000).

Research on the preclinical efficacy of nicotinamide in rodent models of TBI has also resulted in very favorable outcomes (Hoane et al., 2003, Hoane et al., 2006b). Administration of nicotinamide following bilateral, frontal CCI significantly reduced the behavioral impairments and improved recovery of function following injury (Hoane et al., 2003). Administration of nicotinamide (500 mg/kg, ip), 15 min following CCI, reduced the behavioral impairments on the bilateral tactile adhesive removal test; nicotinamide-treated animals were indistinguishable from sham animals (Hoane et al., 2003). Nicotinamide significantly improved the acquisition of reference and working memory tasks in the Morris water maze (MWM). Nicotinamide reduced the size of the injury cavity and the proliferation of glial fibrillary acid protein (GFAP) expression around the lesion cavity 35 days after injury. Using the fluid percussion injury (FPI) model of TBI we have recently shown that nicotinamide at doses of 500 or 50 mg/kg significantly facilitated recovery on the vibrissae-forelimb placing, bilateral tactile adhesive removal, and beam walking tests compared to saline treatment following injury (Hoane et al., 2006b). In fact, the saline-treated animals showed chronic impairments on all 3 tests; while both the 500 and 50 mg/kg doses showed almost total recovery. On the cognitive tests the 500 mg/kg dose, but not the 50 mg/kg dose, improved performance on a working memory task in the MWM. Serum analysis of nicotinamide levels showed that the 500 mg/kg dose significantly elevated concentrations by 30-fold and that the 50 mg/kg dose elevated concentrations 3-fold. Thus, raising nicotinamide concentrations resulted in the significant reduction of the behavioral impairments following FPI (Hoane et al., 2006b). Although nicotinamide has very robust effects on recovery of function, little is known about its mechanisms of action following TBI.

Nicotinamide is a “vita-nutrient” compound and is the amide form of niacin, a soluble B group vitamin, which refers to both nicotinic acid and nicotinamide, which have the same biological activity. A recent review of the medicinal chemistry of nicotinamide has described its complicated biochemistry and unique contributions to treating disease (Yang et al., 2002a). Nicotinamide has two derivatives, nicotinamide adenine dinucleotide (NAD+) and nicotinamide adenine dinucleotide phosphate (NADP), which are coenzymes that are vital to many oxidation-reduction reactions vital to cell metabolism. NAD+ is a precursor for ATP; thus, NAD+ increases neuronal ATP concentration. It has been suggested that nicotinamide prevents depletion of NAD+, thus protecting against ATP depletion and increasing neuroprotection post-injury (Ayoub et al., 1999). In fact, a recent study has shown that systemic administration of nicotinamide elevates cerebral concentrations of ATP and NAD (Yang et al., 2002c).

Nicotinamide is an inhibitor of poly-ADP-ribose polymerase (PARP). PARP is an enzyme found in the nuclei of cells and functions to sense DNA damage, bind to the damaged DNA, catalyze the cleavage of NAD+ into nicotinamide and ADP-ribose, and then use the ADP-ribose to synthesize polymers which attach to nuclear acceptor proteins (Virag and Szabo, 2002). Known as the death substrate, PARP-1 has been identified as a substrate of caspase-7 and caspase-3, which are known as the main executioners of apoptosis (Kaufmann and Kaufman, 1993, Raghupathi et al., 2000, Tewari and Dixit, 1995). Nicotinamide has also been shown to successfully inhibit PARP production (Yang et al., 2002b). Given the potential ability of nicotinamide to reduce cell death, it is likely that this would account for the improved functional outcome following brain injury and may result in reducing many of the secondary pathophysiological events following injury.

Given the numerous studies that have shown that nicotinamide can reduce injury impairments and improve recovery of function in a variety of rodent models of brain injury, it is necessary to investigate the mechanisms by which this treatment has these actions. There has been no attempt to investigate the effect of nicotinamide on BBB breach or apoptosis following TBI. The reduction of these pathophysiological changes following TBI may be responsible for the beneficial effects on behavior that have been shown. Thus, the purpose of the present experiment was to examine the ability of nicotinamide to reduce BBB breach and apoptosis following TBI using a standard model of TBI.

Section snippets

IgG analysis

The initial analysis was a two-factor ANOVA, including treatment (saline or nicotinamide) and sacrifice interval (5 h, 24 h, or 72 h) as factors in the analysis. There were significant main effects for treatment [F(1,28) = 35.26, p < 0.001] and sacrifice interval [F(2,28) = 18.42, p < 0.001], as well as a significant treatment × sacrifice interval [F(2,28) = 6.78, p < 0.004].

Post hoc analysis was performed on the total number of IgG+ cells analyzed by a one-factor ANOVA, including treatment (saline or

Discussion

The results of this study showed that administration of nicotinamide following TBI significantly reduced the breakdown of the BBB. More specifically, the data from the cell count analysis showed that administration of nicotinamide significantly reduced the magnitude of the BBB breakdown at 5, 24, and 72 h following injury.

The entire tissue sections from the rats sacrificed at 5 h post-CCI appeared to have extensive reactivity for IgG, whereas the tissue sections from the rats sacrificed at 24

Subjects

Forty male Sprague–Dawley rats, approximately 3 months old, were used for this experiment. All experimental procedures were reviewed and approved by the Institutional Animal Care and Use Committee and the study was conducted in a facility certified by the American Association for the Accreditation of Laboratory Animal Care. Rats were maintained on a standard 12-h light/dark cycle with food and water available ad libitum.

Surgery

The surgical procedure was performed using aseptic procedures and

Acknowledgment

Research supported by an AREA grant (R15) from NINDS (NS045647-03) to MRH.

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