Traumatic brain injury (TBI)

Original article

The cysteine-rich whey protein supplement, Immunocal®, preserves brain glutathione and improves cognitive, motor, and histopathological indices of traumatic brain injury in a mouse model of controlled cortical impact

Author links open overlay panelElizabeth Ignowski ^a 1, Aimee N. Winter ^a 1, Nathan Duval ^b, Holly Fleming ^b, Tyler Wallace ^a, Evan Manning ^a, Lilia Koza ^a, Kendra Huber ^c, Natalie J. Serkova ^c, Daniel A. Linseman ^d

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Highlights

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Immunocal® significantly enhanced resilience to TBI induced by closed head injury in mice.

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Immunocal® preserved brain GSH/GSSG and attenuated demyelination and neuronal degeneration.

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Most significantly, Immunocal® improved motor and cognitive deficits induced by TBI.

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Immunocal® holds significant promise as a preventative agent for TBI.

Abstract

Traumatic brain injury (TBI) is a major public health problem estimated to affect nearly 1.7 million people in the United States annually. Due to the often debilitating effects of TBI, novel preventative agents are highly desirable for at risk populations. Here, we tested a whey protein supplement, Immunocal®, for its potential to enhance resilience to TBI. Immunocal® is a non-denatured whey protein preparation which has been shown to act as a cysteine delivery system to increase levels of the essential antioxidant glutathione (GSH). Twice daily oral supplementation of CD1 mice with Immunocal^® for 28 days prior to receiving a moderate TBI prevented an ~ 25% reduction in brain GSH/GSSG observed in untreated TBI mice. Immunocal® had no significant effect on the primary mechanical injury induced by TBI, as assessed by MRI, changes in Tau phosphorylation, and righting reflex time or apnea. However, pre-injury supplementation with Immunocal® resulted in statistically significant improvements in motor function (beam walk and rotarod) and cognitive function (Barnes maze). We also observed a significant preservation of corpus callosum width (axonal myelination), a significant decrease in degenerating neurons, a reduction in Iba1 (microglial marker), decreased lipid peroxidation, and preservation of brain-derived neurotrophic factor (BDNF) in the brains of Immunocal®-pretreated mice compared to untreated TBI mice. Taken together, these data indicate that pre-injury supplementation with Immunocal® significantly enhances the resilience to TBI induced by a moderate closed head injury in mice. We conclude that Immunocal® may hold significant promise as a preventative agent for TBI, particularly in certain high risk populations such as athletes and military personnel.

Graphical abstract

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Introduction

Approximately 1.7 million incidences of TBI occur in the United States each year [12]. Of these cases, nearly 75% are categorized as forms of mild TBI including concussions. Chronic traumatic encephalopathy and other forms of dementia have been linked to repetitive mild TBI caused by sports related concussive and subconcussive head trauma in football, hockey, soccer, and wrestling [17], [21], [34], [9]. In a similar manner, blast-related TBI which is estimated to affect 10–20% of veterans returning from the wars in Iraq and Afghanistan, is also associated with an increased risk of chronic traumatic encephalopathy and other types of dementia, as well as posttraumatic stress disorder [11], [18]. Regardless of the cause or severity of TBI, even mild TBI appears to be a significant risk factor for development of dementia including Alzheimer’s disease [15], [16], [25], [29], [43]. Thus, identification of new strategies to enhance resilience against TBI is of particular importance to people participating in “high risk” occupations, such as athletes or military personnel.

Oxidative and nitrosative stress are key elements of the secondary injury processes following TBI [1], [4]. GSH is an essential antioxidant that works in concert with GSH peroxidases, GSH transferases, and peroxiredoxins to detoxify hydroperoxides and other electrophilic species produced during periods of oxidative and nitrosative stress. Several studies suggest that endogenous GSH plays an important protective role against TBI. Brain GSH levels are significantly reduced following TBI induced by controlled cortical impact in rats [47]. Genetic variations in the activity of glutathione-S-transferase-4, a GSH-dependent enzyme that reduces 4-hydroxynonenal, is a determining factor in the extent of neurodegeneration after TBI [2]. Moreover, mice homozygous for deletion of glutathione peroxidase-1 display enhanced susceptibility to brain mitochondrial dysfunction induced by TBI [50]. These findings suggest that a strategy aimed at sustaining or enhancing brain GSH levels may be a viable approach to mitigate secondary injury and the subsequent long term cognitive, physical, and emotional deficiencies induced by TBI.

Along these lines, several studies have shown that administration of the GSH precursor, N-acetylcysteine, just prior to or immediately after TBI, significantly preserved brain tissue and mitochondrial GSH levels, reduced oxidative damage, and preserved neuronal survival [24], [49]. Similarly, treatment with gamma-glutamylcysteine ethyl ester reduced indices of oxidative and nitrosative stress and preserved blood-brain-barrier (BBB) function when given immediately post-TBI [31], [40]. Finally, the GSH analog, S-nitrosoglutathione, decreased BBB disruption, minimized neuronal loss, and increased the expression of neurotrophic factors when administered post-TBI to rats subjected to controlled cortical impact [26], [27] Each of these results indicates the potential of enhancing GSH as a therapeutic approach for TBI. Unfortunately, few of these previous studies evaluated the effects of GSH precursor supplementation on cognitive or motor deficits induced by TBI and as a result, it is presently unclear what therapeutic benefit this strategy might realistically hold for patients suffering from TBI.

The nutritional supplement, Immunocal®, is a non-denatured whey protein designed to augment the available intracellular GSH pool. Cellular GSH concentrations are highly dependent on the availability of cysteine, which is the limiting precursor in GSH synthesis [36], [46]. The cysteine precursor, cystine, occurs at high levels in Immunocal®, as does the direct GSH precursor, glutamylcysteine [6], [7]. Immunocal® has been shown to significantly increase blood or lymphocyte GSH levels in HIV-seropositive or cystic fibrosis patients, respectively [8], [19]. Immunocal® is one of only a handful of nutritional supplements that are included in the Physician’s Desk Reference and is comprised of natural food protein placing it in the FDA category of generally recognized as safe [39]. We have recently found that Immunocal® preserves blood and spinal cord GSH levels and delays disease onset in a transgenic mouse model of amyotrophic lateral sclerosis [41]. Similarly, Immunocal® restored GSH homeostasis in the CNS and ameliorated behavioral deficits in a mouse model of schizophrenia [45]. Based on the above findings, we hypothesized that supplementation with Immunocal® prior to TBI in mice would provide enhanced resilience against oxidative damage, neuronal cell death, and cognitive and motor impairments induced by a closed head impact injury.

Section snippets

Animal care and treatment

All animal work was conducted under a protocol approved by the University of Denver Institutional Animal Care and Use Committee. Male CD1 Elite mice (35 days-old) were purchased from Charles River Laboratories (Hollister, CA). Mice received a numbered ear tag upon arrival for identification purposes, and then were allowed one week to acclimate to the animal facility at the University of Denver before beginning the study. Mice were then randomly assigned and evenly distributed among one of three 

Pre-injury supplementation with Immunocal® does not affect the primary mechanical injury induced by a moderate TBI

Throughout the study, mice were equally divided into the following three groups: Group 1, Sham surgery controls; Group 2, untreated TBI mice; and Group 3, mice pretreated with Immunocal® for 28 days prior to TBI. The extent of brain injury was initially assessed at 72 h post-TBI by MRI analysis of BBB disruption. In mice subjected to TBI, T1 weighted images taken with gadolinium contrast showed areas of hyper-intensity which indicate BBB disruption (Fig. 1A and B, see asterisk in the TBI image

Discussion

The pathophysiological processes underlying the short and long term injury sequelae associated with TBI are complex. The primary injury is mechanical, resulting from an external force, and leads to tissue deformation, tearing of blood vessels and neuronal axons, necrotic cell death, and initiation of secondary injury processes. Secondary injury mechanisms may include intracranial hemorrhage, excitotoxicity, ionic disturbances, decreased cerebral blood flow, edema, inflammation, mitochondrial

Acknowledgments

None.

Funding

This study was supported in large part by funding from Immunotec Inc. (Quebec, Canada), which is the manufacturer of Immunocal®. Funding was also provided by a pilot grant from the Knoebel Institute for Healthy Aging at the University of Denver. The University of Colorado Animal Imaging Shared Resources are supported by the University of Colorado Cancer Center (NCI P30 CA046934) and the Colorado Clinical and Translational Sciences Institute (NIH/NCATS UL1 TR001082).

Conflicts of interest

The authors have received

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