Elsevier

Experimental Neurology

Volume 183, Issue 2, October 2003, Pages 406-417
Experimental Neurology

Regular article
Proliferation and neuronal differentiation of mitotically active cells following traumatic brain injury

https://doi.org/10.1016/S0014-4886(03)00241-3Get rights and content

Abstract

Recent studies have identified endogenous neural stem cells in adult rodent brains. The present study characterizes the early response of mitotically active cells in the brain to traumatic brain injury. Animals were subjected to lateral fluid percussion injury and sacrificed at various times after injury. To examine increases in cell proliferation animals were injected with the mitotic marker bromodeoxyuridine (BrdU) 24 h before sacrifice. Increased numbers of mitotically active cells were observed at 2 days in the subgranular zone (SGZ) and the subependymal zone (SEZ) under the injury site. To characterize the differentiation potential of these cells, animals were injected with BrdU 18 and 20 h after injury, then sacrificed at multiple time points after injury. Histologically, co-localization with βIII-tubulin (neuronal marker) and BrdU was evident at 10 and 15 days postinjury in the SGZ. Flow cytometry analysis was used to quantitatively assess neurogenesis in the SEZ. Animals were sacrificed 1, 5, or 10 days after injury and tissue sections extracted, grown in tissue culture for 24 h, fixed, and stained for nestin and βIII-tubulin to identify newly formed neurons. The percentage of cells expressing both markers was determined using flow cytometry analysis. There was a significant increase in newly differentiated neurons by 10 days postinjury in the SEZ. Thus, we conclude that traumatic brain injury stimulates an increase in proliferation of endogenous neural stem/progenitor cells and that a significant number of these express a neuronal marker. This response may be the brain’s way of trying to heal itself after injury.

Introduction

Traumatic brain injury (TBI) is the leading cause of death in individuals under 25 years of age (Thurman and Guerro, 1999). More than 100,000 people die each year from brain trauma and another 50,000 are permanently disabled. Even individuals with only a moderate injury often have a long-lasting disruption of their way of life (Hilton, 1994). Brain trauma is a multifaceted injury with mechanical, ischemic, and excitotoxic components (McIntosh et al., 1996 review). Following the initial mechanical insult, secondary pathways are activated that contribute to ischemic and excitotoxic damage Kawamata et al 1995, Xiong et al 1997, Azbill et al 1997. At present, therapies are aimed at minimizing the secondary damage by interventions in very narrow windows of opportunity. However, there is no cure for the initial mechanical damage or the secondary damage that is not avoided.

Recently, in contrast to the dogma, neural stem cells have been identified in adult mammals that, have the potential to differentiate into either glial or neuronal phenotypes Richards et al 1992, Reynolds and Weiss 1992; Lois and Alvarez-Buylla 1993, Kuhn et al 1996, Laywell et al 1999. There are two locations in the adult rodent brain that have been identified as proliferation zones in adult animals. One of these locations is the subependymal zone (SEZ) or subventricular zone of the lateral ventricles. In rodents, the neural stem cells in the SEZ normally function to migrate down the rostral migratory pathway and replace olfactory neurons (Lois and Alverez-Buylla, 1994). The second location is the subgranular zone of the hippocampal formation (SGZ) at the dentate gyrus (DG)–hilus interface. These cells normally migrate into the DG and become granule cells Kuhn et al 1996, Parent et al 1997. Thus a constant slow rate of neurogenesis is occurring all the time in adult animals.

Following insults to the brain such as seizures and ischemia, there is an increase in the number of mitotically active cells in the brain. In these two models, an upregulation of dividing cells is observed in the SGZ Parent et al 1997, Parent et al 1999, Parent et al 2002, Liu et al 1998, Takagi et al 1999, Nakagawa et al 2000, Jin et al 2001, Levison et al 2001, Iwai et al 2002, Takasawa et al 2002). These cells migrate into the granule cell layer and differentiate into granule cell neurons. At present little is known about the response of endogenous neural stem/progenitor cells following traumatic brain injury Dash et al 2001, Kernie et al 2001, Chirumamilla et al 2002, Braun et al 2002. Injection of bromodeoxyuridine (BrdU) into the brain parenchyma as a stab injury model revealed an increased BrdU immunoreactivity in the subependymal zone both ipsilaterally and contralaterally, while there was an increase in macrophages/microglia around the needle track (Tzeng and Wu, 1999). Recent studies using the controlled cortical impact (CCI) model of TBI have demonstrated an increase in neurogenesis in the DG/SGZ Dash et al 2001, Kernie et al 2001. In the lateral fluid percussion injury (LFPI) model, it has been shown that there is an early proliferation response; the majority of the cells labeled with the mitotic marker also expressed an astrocytic marker or a macrophage marker (Chirumamilla et al., 2002). In the present report we are characterizing a time course of the proliferation response of neural progenitor cells in the brain to lateral fluid percussion injury, and the extent to which neuronal differentiation is occurring in these cells stimulated to divide by injury.

Section snippets

Materials and methods

Adult Sprague–Dawley (Harlan, Indianapolis, IN, USA) rats, 300–350 g, were used in accordance with protocols approved by the Institutional Animal Care and Use Committee. All efforts were made to minimize pain and suffering to the animals and to minimize the number of animals required.

Proliferation study: histology

Animals were injected with the mitotic marker BrdU at various time points after injury and sacrificed 24 h later to determine changes in the proliferation rate after injury. Figure 1A depicts the typical pattern of BrdU-IR cells in an animal sacrificed 2 days after injury. BrdU-IR cells are visible throughout the white matter tracts lining the ipsilateral wall of the lateral ventricle and extending into the corpus callosum. There are also BrdU-IR cells in the cortex under the injury and in the

Discussion

We have presented data demonstrating an increase in mitotically active cells in the SGZ and SEZ, the neural stem cell proliferation zones in the brain, following lateral fluid percussion injury in adult animals, both ipsi- and contralaterally. There are two waves of increased proliferation in the SGZ, at 2 and 8 days postinjury. These cells degrade or migrate away over the 2 weeks studied. Co-localization of the mitotic marker with a neuronal marker is depicted histologically. There was not a

Acknowledgements

This work was supported by NIH Grant NS12587, Commonwealth Neurotrauma Initiative Grant 02-319, and the Reynolds and Lind Lawrence Foundations to M.R.B. and NIH Grant P30CA16059 to the Massey Cancer Center.

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