Elsevier

Brain Research

Volume 1065, Issues 1–2, 14 December 2005, Pages 8-19
Brain Research

Research Report
Caspase-mediated cell death predominates following engraftment of neural progenitor cells into traumatically injured rat brain

https://doi.org/10.1016/j.brainres.2005.09.059Get rights and content

Abstract

Neural progenitor cells (NPCs) have been shown to be a promising therapy for cell replacement and gene transfer in neurological diseases including traumatic brain injury (TBI). However, NPCs often survive poorly after transplantation despite immunosuppression, and the mechanisms of graft cell death are unknown. In this study, we evaluated caspase- and calpain-mediated mechanisms of cell death of neonatal mouse C17.2 progenitor cells, transplanted at 24 h following lateral fluid percussion brain injury (FP) in rats. Adult Male Sprague–Dawley rats (n = 30) were subjected to lateral FP injury (n = 18) or sham surgery (n = 12). C17.2 cells labeled with green fluorescent dye (CMFDA) were engrafted in the perilesional deep cortex, and animals were sacrificed at 24 h, 72 h and 1 week post-transplantation. Pro-apoptotic caspase-mediated cleavage products (Ab246) and calpain-mediated cleavage products (Ab38) were detected in the engrafted cells using immunohistochemistry. Only 2 to 4.5% of grafted NPCs were found to survive at 24 h post-transplantation, regardless of injury status of the host brain, although brain-injured animals had significantly fewer graft cells than sham-injured animals. Limited caspase and calpain-mediated graft cell death was observed in both sham- and brain-injured animals, and caspase-mediated graft cell death was significantly greater than calpain-mediated graft cell death in all animals. Brain-injured animals had significantly increased caspase-mediated graft cell death compared to sham-injured animals. These results suggest that both the caspase and calpain family of proteases are involved in graft cell death, and that caspase-mediated apoptotic graft cell death predominates in the acute post-traumatic period following TBI.

Introduction

There is increasing interest in the use of neural progenitor cells (NPCs) as a therapeutic tool in various neurological disorders including traumatic brain injury (TBI) [66]. C17.2 cells are a clonal multipotent NPC line derived from neonatal mouse cerebellar external granular layer immortalized by the retrovirus-mediated transduction of avian v-myc oncogene [63]. These cells have been shown to survive, engraft into the host architecture following transplantation into newborn or adult murine brain [71] and are capable of differentiating into cells of neuronal and glial lineage [60], [72]. Following transplantation of C17.2 cells into rodent brains subjected to ischemia or TBI, some cells are seen to engraft and improve neurological outcome [36], [56]. However, NPCs have been found to have limited survival, when engrafted into the injured central nervous system (CNS) following TBI, with only 30%–70% of animals having surviving graft cells between 2 and 12 weeks, with or without immunosuppression [11], [56]. The mechanism of this acute graft cell death of NPCs in TBI remains unclear.

In vitro and in vivo transplant studies in rodent models of neurodegeneration have shown that transplanted embryonic or fetal tissue undergoes both necrotic and apoptotic death [12], [18], [25], [26], [42], with a critical window of 4 to 7 days post-implantation, during which an estimated 90–95% of grafted embryonic cells die [7], [75]. Apoptosis is a genetically programmed type of active cell death involving gene transcription and protein synthesis in which the cell uses specialized cellular machinery to kill itself, thereby promoting non-immunogenic elimination of abnormal cells during CNS development and aging [9], [29]. Apoptosis is a complex process involving a variety of different signaling pathways including caspase-dependent intrinsic and extrinsic pathways [4], [54], [67] and caspase-independent pathways [33], [70], [77]. Caspases are synthesized and exist mostly in the cytoplasm of viable cells as an inactive pro-enzyme and once activated cleaves a variety of molecules such as poly ADP-ribose polymerase (PARP), protein kinase, spectrin, actin and DNA-dependent protein kinase [39], [90].

Calpains belong to another family of proteases and are activated predominantly by increased intracellular Ca2+, phosphorylation and proteolysis of cytoskeletal proteins. Calpains are localized to multiple locations within the cell, including in some cases to focal adhesion molecules. Activated calpain cleaves various cytoskeletal proteins such as spectrin, tubulin, microtubule-associated proteins and neurofilament proteins [40], [69] and along with cathepsins have been implicated in both neuronal necrosis and apoptosis [46], [51], [55]. Interestingly, there appears to be cross-talk between the calpain and caspase-dependent pathways [10], [39], [47], [52], [53], and both proteins have many common substrates including cytoskeletal and regulatory proteins [16]. Both calpain and caspase-3 activation induces proteolysis of the cytoskeletal protein α-spectrin, upstream of endonuclease DNA fragmentation [52]. Caspases and calpains have been implicated in embryonic and fetal graft cell death following brain transplantation into normal and Parkinson rodent models [18], [26], [43], [89].

Cell death in TBI involves a phenotypic spectrum ranging from necrotic to apoptotic depending on the extent of injury, brain region, time post-injury and the activation/release of a complex cascade of secondary factors including excitotoxic neurotransmitters, reactive oxygen species, inflammatory cells and molecules [19], [21], [22], [38], [50], [58], [68], [86], [73]. Multiple caspases and calpains have been shown to be activated after TBI [8], [20], [37], [57], [64], [76], and transplantation of NPCs into the injured brain could expose cell transplants to these death-promoting cascades.

We hypothesized that NPCs used for therapy in TBI can be subjected to both caspase and calpain-mediated cell death, and that the hostile post-traumatic environment of TBI may aggravate graft cell death. The current study is novel in that we evaluated the mechanisms underlying cell death of NPCs xeno-grafted into sham and traumatically injured brains of rodents. This information is crucial to provide fundamental insights into prolonging graft cell survival following transplantation.

Section snippets

Cell preparation techniques for transplantation procedure

The C17.2 NPCs were maintained in plastic culture flasks in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum and 5% horse serum along with 2 mM glutamine. The cells were cultured in a humidified atmosphere of 5% CO2 and 95% air at 37 °C. The cells were passaged at 90% confluence, and for transplantation, 90% confluent cultures were used. The cells were trypsinized, detached, spun down and suspended in Hank's balanced salt solution (HBSS) at a concentration of

Graft morphology

All the animals receiving NPC transplants showed surviving graft cells. On microscopic examination, a small degree of hemorrhage surrounding the needle tract was observed in all animals. The transplanted green fluorescent C17.2 cells possessed a rounded morphology at 24 h post-implantation and seemed to attain a bipolar morphology by 72 h (Fig. 2, Fig. 3). The graft cells remained localized to the site of implantation at all the three time points (Fig. 2, Fig. 3). Ab246 and Ab38 staining for

Discussion

The present study is the first to evaluate the mechanisms underlying NPC death following transplantation into the traumatically injured brain and to evaluate the potential involvement of the activated proteases caspsase-3 and calpains in the death of NPCs. We have shown that NPCs undergo both caspase- and calpain-mediated death following transplantation into injured cortex, as demonstrated by an upregulation of caspase- and calpain-specific α-spectrin degradation products in the cells. We have

Acknowledgments

These studies were supported, in part, by NIH NS 40978 and a Veterans Administration Merit Review grant. We would like to thank Rishi Puri, Jamie Plevy and Diego Morales for the technical support.

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