The role of matrix metalloproteinases in infant traumatic brain injury

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Abstract

Matrix metalloproteinases (MMPs) play an essential role in tissue repair, cell death and morphogenesis and may constitute therapeutic targets for acute brain injuries. In this study, we investigated the expression of 72 kDa and 92 kDa collagenases MMP-2 and MMP-9 at transcriptional, functional and protein expression level following traumatic brain injury in infant rats. Seven-day-old Wistar rats were subjected to head trauma using a weight drop device. Pups were sacrificed at defined time points (2–72 h) after trauma and brains were processed for molecular studies (semiquantitative and real-time PCR, Western blot, gelatin zymography) and histology. Trauma triggered widespread cell death in the cortex, basal ganglia and white matter. mRNA levels for MMP-2 and -9 were increased in the brain at 12–72 h after trauma. Protein expression of the analyzed MMPs and activity of MMP-2 were increased at 12 h and peaked at 24 h after trauma. Intraperitoneal injection of GM6001 (Ilomastat), an MMP inhibitor, 2 h after trauma, substantially attenuated traumatic brain injury in a dose-dependent manner. These findings causally link the MMPs to trauma-induced neuronal cell death in the immature rodent brain. MMPs might serve as useful targets for therapeutic approaches aimed at preserving neuronal function in the immature brain in the context of mechanical injury.

Introduction

Traumatic brain injury (TBI) constitutes a major cause of morbidity and mortality in the industrialized world (Goldstein, 1990, Sosin et al., 1995, Thurman et al., 1999). Clinical studies suggest that age decidedly influences morbidity and mortality after head injury in children, with those under 4 years of age showing worst outcomes (Adelson and Kochanek, 1998, Koskiniemi et al., 1995). Differences in the mechanisms by which traumatic brain injury was sustained and the higher incidence of non-accidental closed head traumata in the very young (James, 1999, Kraus et al., 1987) may partly account for these findings. In addition, experimental studies suggest that the developing brain may be more prone to suffering irreversible neuronal loss following traumatic injury (Bittigau et al., 1999), but the mechanisms involved are only partly understood.

Matrix metalloproteinases (MMPs) are zinc-endopeptidases with multifactorial actions in central nervous system (CNS) physiology and pathology that are collectively able to degrade or modify components of the extracellular matrix (Nagase and Woessner, 1999). Target substrates include collagens, gelatin, fibronectin, laminin, elastin and proteoglycans. The biological activity of MMPs is strictly regulated via gene transcription, proenzyme activation, and dynamic inhibition by tissue inhibitors of metalloproteinases (TIMPs) (Yong, 2005, Vu et al., 1998). MMPs participate in important physiological processes including embryological remodeling, wound healing, angiogenesis, bone remodeling, ovulation, and implantation (Yong, 2005, Vu et al., 1998).

Experimental work has shown that overactivity of MMPs is involved in disease processes of the central nervous system (CNS), such as multiple sclerosis and Alzheimer’s disease (Yong, 2005, Lukashev and Werb, 1998, Yong et al., 1998). Evidence is also accumulating implicating involvement of MMPs in acute brain injury following stroke and trauma. MMP activity is upregulated after cerebral ischemia and edema (Morita-Fijimura et al., 1999), and MMPs are involved in stroke pathophysiology (Romanic et al., 1998, Rosenberg et al., 1996, Gasche et al., 1999, Heo et al., 1999). In stroke patients, MMP-9 biomarkers are correlated with clinical outcomes (Montaner et al., 2001, Montaner et al., 2003).

By degrading neurovascular matrix, MMPs promote injury of the blood–brain barrier, edema and hemorrhage (Asahi et al., 2001, Wang et al., 2003, Lo et al., 2003, Shigemori et al., 2006). By disrupting cell–matrix signaling and homeostasis, MMPs trigger neuronal and glial cell death (Lee and Lo, 2004, Gu et al., 2002).

In traumatic brain injury (TBI) models, it has been shown that activity of MMPs is increased after TBI (Wang et al., 2000) or spinal cord injury (Noble et al., 2002). Furthermore, MMP-9 knock-out mice showed fewer motor deficits and smaller contusion volumes compared with wild-type mice (Wang et al., 2000). In spinal cord injury, MMP-9 null mice exhibited significantly less disruption of the blood–spinal cord barrier, attenuation of neurotrophil infiltration and significantly better locomotor recovery (Noble et al., 2002). Hence, there is a movement towards the development of MMP inhibitors for the treatment of acute brain injuries.

In this study, we investigated the hypothesis that MMP-2 and MMP-9 may be involved in the pathophysiology of neuronal damage after traumatic injury in the developing brain. We tested this hypothesis by performing experiments using a weight drop model of traumatic brain injury in infant rats. Previously, we provided detailed description of the distribution pattern and time course of the neurodegenerative response to brain trauma in 7-day-old rats. We have performed detailed analysis of the delayed neurodegenerative response to trauma by electron microscopy, which has confirmed that neurons which degenerate in a disseminated and delayed fashion fulfil ultrastructural criteria for apoptosis (Bittigau et al., 1999). In addition, we have reported that marked activation of caspase-3 occurs in affected brain regions. This previously presented morphological and biochemical evidence represents the basis for our assumption that, in the study outlined here, we have been investigating mechanisms pertaining to apoptotic neurodegeneration following trauma to the developing rat brain. Our findings implicate involvement of MMP-2 and MMP-9 in the pathophysiology of TBI in the developing rat brain and suggest that MMPs may constitute useful therapeutic target to ameliorate cell loss following TBI in children.

Section snippets

Animal experiments

All animal experiments were performed in accordance with the guidelines of the Technical University in Dresden, Germany.

Trauma

Seven-day-old Wistar rat pups (Charles River, Sulzfeld, Germany), weighing 13–16 g, were anesthetized with halothane (induced with 4% and maintained with 1.5% in balanced room air) and subjected to head trauma as previously described (Bittigau et al., 1999, Pohl et al., 1999). Following fixation of the animals’ head, a skin incision was made to expose the skull. The trauma

Results

Rats subjected to sham surgery (n = 9) or trauma (n = 30) recovered within 10 min after anesthesia. At 24 h following percussion head trauma in 7-day-old rats, widespread cell death was detected by silver, TUNEL or FluoroJade staining in the frontal, parietal, cingulate and retrosplenial cortex, the thalamus, the dentate gyrus, the subiculum and the striatum (Fig. 1).

Discussion

Trauma triggers widespread and diffuse apoptosis in the immature rat brain in an age-dependent manner. Peak sensitivity is observed at 3 to 7 postnatal days. This vulnerable period corresponds to the brain growth spurt of rats and mice (Dobbing and Sands, 1979). Mainly affected brain regions are cerebral cortex, thalamus and caudate nucleus. The apoptotic nature of trauma-induced cell death has been previously confirmed by a combination of histological, immunohistochemical and molecular studies

Acknowledgment

Supported by DFG grant IK2/5-1.

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