Introduction Methods (continued)WT & tPA KO

Contusion, 50 kdynes

T8-T10Ventral

Perfusion at 1,7, 22 dpi

Iba-1 staining, CNP, Caspase-3

WT 1 dpi

Rostral

Iba-1 DAPI

Acknowledgements Dr. Stella Tsirka and the Tsirka Lab members AGEP is funded by NSF grant # 0450106Jonathan Patete and Jaqueline M. Smith

7dpi WT

7dpi tPA-KO

1 μg/ mL

10 μg/ mL

100 μg/ mL

22 dpi WT

22 dpi tPA- KO

Caspase-3 DAPI Merged

CNP DAPI Merged

In culture, one source of microglia activation is exposure to foreign particles. To determine the potential toxicity and reaction of microglia to iron oxide nanoparticles. Applications of nanoparticles include terabit magnetic storage devices, catalysis, sensors and MRI for diagnosis and theraputics.We hope to use nanoparticles to track microglia in vivo using magnetic resonance imagining.  The results of this experiment will advance our knowledge on the many practical applications of nanosized iron oxide particles.

N9 microglia were treated with different concentrations of rombohedral Iron oxide nanoparticles and monitored for five days. Iba-1 staining and PI staining were performed to observe microglial activation and cell death in response to the foreign particles exposure.

•staining for caspase, and CNP has to be optimized to determine relationship between microglia activation and oligodendrocyte apoptosis •decrease in microglia activation in the absence of tPA from figure 1.

•iron oxide nanoparticles engulfed and non-toxic in vitro.• Electron microscopy to determine how many engulfed by microglia •MRI to view microglia in vivo to better understand the mechanisms involved with microglia activation after spinal cord injury.

Following spinal cord injury, tPA levels increase dramatically at the injury site and its binding to the protein surface receptor annexin II in microglia causes rampant activation. Activated microglia become phagocytic and secrete cytotoxic factors, such as hydrogen peroxide, to fight off infection. Additionally, they release pro-inflammatory molecules that encourage migration and proliferation of more activated microglia to the site of injury. Changes in their gene expression cause upregulation of cytokines that may cause excitotoxic death in neurons and oligodendrocytes . As a consequence of oligodendrocyte death, the protective myelin sheath found around axons is destroyed, leading to the permanently dehabilitating effects characteristic of spinal cord injury. We hypothesize that tPA deficiency leads to decreased microglial activation, decreased demyelination and decreased numbers of apoptotic oligodendrocytes in return.

Results

Caudal

ConclusionstPA KO 1dpi

Rostral

Rostral

Rostral

Rostral

Rostral

Caudal

Caudal

Caudal

Caudal

Caudal

Caudal

Figure 1

Figure 2

Figure 3 Untreated

Representative bright field and florescent microscope images of N9 microglia after exposure to various concentrations of nanoparticles.

representative confocal images of spinal cord sections from wild-type and tPA-KO mice stained for Iba-1 at various time points post injury

Spinal cord tissue sections stained for oligodendrocytes with cyclic nucleotide phosphodiesterase and apoptosis with caspase-3.

Tsirka, S. E. (2002). Tissue Plasminogen Activator as a modulator of Neuronal Survival and Function. Biochemical Society Transactions, 30(2). Gravanis, I., & Tsirka, S. E. (2004, October 19). Tissue Plasminogen Activator and Glial Function. Wiley InterScience, 49, 177-183. Thorek, D. J., Chen, A. K., Czupryna, J., & Tourskas, A. (2006, February 16). Superparamagnetic Iron Oxide Nanoparticle Probes for Molecular Imaging. Annals of Biomedical Engineering, 34(1), 23-33.

References

Iba-1 DAPI

Methods