Substantia nigra vulnerability after a single moderate diffuse brain injury in the rat

https://doi.org/10.1016/j.expneurol.2011.12.003Get rights and content

Abstract

Dementia and parkinsonism are late-onset symptoms associated with repetitive head injury, as documented in multiple contact-sport athletes. Clinical symptomatology is the likely phenotype of chronic degeneration and circuit disruption in the substantia nigra (SN). To investigate the initiating neuropathology, we hypothesize that a single diffuse brain injury is sufficient to initiate SN neuropathology including neuronal loss, vascular disruption and microglial activation, contributing to neurodegeneration and altered dopamine regulation. Adult, male Sprague–Dawley rats were subjected to sham or moderate midline fluid percussion brain injury. Stereological estimates indicated a significant 44% loss of the estimated total neuron number in the SN at 28-days post-injury, without atrophy of neuronal nuclear volumes, including 25% loss of tyrosine hydroxylase positive neurons by 28-days post-injury. Multi-focal vascular compromise occurred 1–2 days post-injury, with ensuing microglial activation (significant 40% increase at 4-days). Neurodegeneration (silver-stain technique) encompassed on average 21% of the SN by 7-days post-injury and increased to 29% by 28-days compared to sham (1%). Whole tissue SN, but not striatum, dopamine metabolism was altered at 28-days post-injury, without appreciable gene or protein changes in dopamine synthesis or regulation elements. Together, single moderate diffuse brain injury resulted in SN neurovascular pathology potentially associated with neuroinflammation or dopamine dysregulation. Compensatory mechanisms may preserve dopamine signaling acutely, but subsequent SN damage with aging or additional injury may expose clinical symptomatology of motor ataxias and dementia.

Highlights

► Substantia nigra (SN) has neurovascular vulnerability to one diffuse brain injury.► Reduction in SN neurons by one month post-injury; most are dopaminergic.► However, dopamine metabolism nor synthesis are compromised in the striatum.► Injury-induced vascular compromise lead to sustained microglial activation in SN.

Introduction

Loss of dopaminergic innervation from the substantia nigra (SN) has been implicated in late onset Parkinson's disease (Bernheimer et al., 1973, Hoehn and Yahr, 1967, Hornykiewicz and Kish, 1987, Lees et al., 2009, Shih et al., 2006). Idiopathic post-mortem pathology shows significant cell dropout and loss of pigmentation among a host of other molecular and cellular markers of cell death (Bernheimer et al., 1973, McGeer et al., 1988). This neuropathology has been modeled using neurotoxic lesions of dopaminergic neurons in the SN in rodents and non-human primates (Bove et al., 2005, Javitch et al., 1985, Smith et al., 1993). Additionally, experimental cerebral ischemia results in SN susceptibility to metabolic dysfunction associated with oxidative damage (Hall et al., 1996). Interestingly, the gender bias for Parkinson's disease weighs heavily toward males, with a world-wide ~ 2:1 bias (Litvan et al., 2007).

Concussive injury occurs in nearly all contact sports, most prominently in boxing and American football. In these athletes, published reports describe the condition colloquially known as ‘punch drunk’ dating back to the 1920's (Martland, 1928), predating cellular and molecular techniques to describe the disease process (Roberts et al., 1990). Contemporary nomenclature defined this condition as the degenerative disease dementia pugilistica, and more recently as chronic traumatic encephalography, where onset often occurs after a career in contact sports with repeated blows to the head (Erlanger et al., 1999, Nowak et al., 2009, Smith et al., 2008, Unterharnscheidt, 1995a, Unterharnscheidt, 1995b, Unterharnscheidt, 1995c, Unterharnscheidt, 1995d, Unterharnscheidt, 1995e). Within the past decade, several studies have reported patients exhibiting parkinsonian symptoms who have participated in contact sports during their lives (Forstl et al., 2010, McKee et al., 2009). The brains of these athletes exhibit decreased tyrosine hydroxylase expression (Yan et al., 2007), chronic traumatic encephalopathy and regional pallor in the SN (Casson et al., 2006, Forstl et al., 2010, Omalu et al., 2005, Omalu et al., 2006, Omalu et al., 2010), among other neuropathological findings across the brain. The gender bias for brain injury lies predominantly with males, accounting for 80–90% of all injuries (Langlois et al., 2004).

Despite the basal ganglia susceptibility to oxidative damage, the majority of previous experimental brain injury studies have overlooked the SN in their results and discussions. Some reports have quantitatively measured the SN as part of an extensive survey of brain regions in rat, but findings were neither discussed nor further explored (Hicks et al., 1996, Hovda et al., 1991, Yoshino et al., 1991). Only recently has the vulnerability of the substantia nigra been investigated after experimental TBI (lateral FPI), where a 15% unilateral loss of dopaminergic neurons by 1 week progresses to 30% loss by 26 weeks, concomitant with microglial activation only at the acute time point (Hutson et al., 2011). Midline fluid percussion injury (mFPI) in rodents provides a clinically relevant model of diffuse brain injury that reproduces features of human pathology (e.g. axonal injury) and ensuing motor and cognitive deficits (Lifshitz, 2008). Using a single mFPI of moderate severity in the rat, we investigate the initiating histopathological vulnerability of the SN and quantify damage to the neurovascular unit with the intent to further understand the etiology of neurological conditions involving SN degeneration.

Section snippets

Midline fluid percussion brain injury (mFPI)

Adult male Sprague–Dawley rats (350–375 g) were subjected to mFPI consistent with methods previously described (Hosseini and Lifshitz, 2009, Lifshitz et al., 2007). Final animal numbers are indicated in the results section for each study. Briefly, rats were anesthetized with 5% isoflurane in 100% O2 and maintained at 2% via nose cone. During surgery, body temperature was maintained with a Deltaphase® isothermal heating pad (Braintree Scientific Inc., Braintree, MA). The animal was secured in a

Neurons

This section reports neuronal damage recorded in the SN in somal, axonal and synaptic compartments after moderate diffuse brain injury.

Discussion

Recent post-mortem studies of American football players have found pallor or paleness of the SN (McKee et al., 2009, Omalu et al., 2005, Omalu et al., 2006, Omalu et al., 2010), prompting us to explore neurovascular pathology in the SN after a single experimental diffuse brain injury. The present study using the mFPI model demonstrates significant decreases in total and TH-positive neuronal cell number and total neuronal density by 28 days post-injury, without change in neuronal nuclear volume.

Acknowledgments

With generous thanks to Amanda Lisembee, Deanne Hopkins, Kelley Hall and Stewart Surgener without whose technical expertise and insightful support this study would not have been possible. Supported, in part, by University of Kentucky College of Medicine, NIH NINDS R01 NS065052, NIH NIA T32 AG000242, NIH NINDS F31 NS067899, Kentucky Spinal Cord and Head Injury Research Trust (KSCHIRT) 7-11, KSCHIRT 11-2a, NIH P50 NS039787 and NIH NINDS P30 NS051220.

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