ABSTRACT
Intervertebral disk degeneration is a progressive and debilitating disease with multifactorial causes. Nitric oxide (NO) might contribute to the cell death pathway. We evaluated the presence of the constitutive form of the neuronal NOS (nNOS) in both health and degenerated intervertebral disk through qPCR and immunohistochemistry. We also analyzed the potential role of nNOS modulation in the tail needle puncture model of intervertebral disk degeneration. Male Wistar rats were submitted to percutaneous disk puncture with a 21-gauge needle of coccygeal vertebras. The selective nNOS pharmacological inhibitor N (ω)-propyl-L-arginine (NPLA) or a nNOS-target siRNA (siRNAnNOShum_4400) was injected immediately after the intervertebral disk puncture with a 30-gauge needle. Signs of disk degeneration were analyzed by in vivo magnetic resonance imaging and histological score. We found that intact intervertebral disks express low levels of nNOS mRNA. Disk injury caused a 4 fold increase in nNOS mRNA content at 5 h post disk lesion. However, NPLA or nNOS-target siRNA slight mitigate the intervertebral disk degenerative progress. Our data show evidence of the nNOS presence in the intervertebral disk and its upregulation during degeneration. Further studies would disclose the nNOS role and its potential therapeutical value in the intervertebral disk degeneration.
Subject(s)
Intervertebral Disc Degeneration/enzymology , Intervertebral Disc/enzymology , Nitric Oxide Synthase Type I/metabolism , Animals , Arginine/analogs & derivatives , Arginine/pharmacology , Cell Line, Tumor , Disease Models, Animal , Enzyme Inhibitors/pharmacology , Gene Knockdown Techniques , Humans , Immunohistochemistry , Intervertebral Disc/diagnostic imaging , Intervertebral Disc/pathology , Intervertebral Disc Degeneration/diagnostic imaging , Intervertebral Disc Degeneration/pathology , Magnetic Resonance Imaging , Male , Nitric Oxide Synthase Type I/antagonists & inhibitors , Nitric Oxide Synthase Type I/genetics , Polymerase Chain Reaction , RNA, Messenger/metabolism , RNA, Small Interfering , Rats, Wistar , Sacrococcygeal RegionABSTRACT
BACKGROUND: Glioblastoma multiforme is the most aggressive brain tumor. Microglia are prominent cells within glioma tissue and play important roles in tumor biology. This work presents an animal model designed for the study of microglial cell morphology in situ during gliomagenesis. It also allows a quantitative morphometrical analysis of microglial cells during their activation by glioma cells. METHODS: The animal model associates the following cell types: 1- mCherry red fluorescent GL261 glioma cells and; 2- EGFP fluorescent microglia, present in the TgH(CX3CR1-EGFP) mouse line. First, mCherry-GL261 glioma cells were implanted in the brain cortex of TgH(CX3CR1-EGFP) mice. Epifluorescence - and confocal laser-scanning microscopy were employed for analysis of fixed tissue sections, whereas two-photon laser-scanning microscopy (2P-LSM) was used to track tumor cells and microglia in the brain of living animals. RESULTS: Implanted mCherry-GL261 cells successfully developed brain tumors. They mimic the aggressive behavior found in human disease, with a rapid increase in size and the presence of secondary tumors apart from the injection site. As tumor grows, mCherry-GL261 cells progressively lost their original shape, adopting a heterogeneous and diffuse morphology at 14-18 d. Soma size increased from 10-52 µm. At this point, we focused on the kinetics of microglial access to glioma tissues. 2P-LSM revealed an intense microgliosis in brain areas already shortly after tumor implantation, i.e. at 30 min. By confocal microscopy, we found clusters of microglial cells around the tumor mass in the first 3 days. Then cells infiltrated the tumor area, where they remained during all the time points studied, from 6-18 days. Microglia in contact with glioma cells also present changes in cell morphology, from a ramified to an amoeboid shape. Cell bodies enlarged from 366 ± 0.0 µm(2), in quiescent microglia, to 1310 ± 146.0 µm(2), and the cell processes became shortened. CONCLUSIONS: The GL261/CX3CR1 mouse model reported here is a valuable tool for imaging of microglial cells during glioma growth, either in fixed tissue sections or living animals. Remarkable advantages are the use of immunocompetent animals and the simplified imaging method without the need of immunohistochemical procedures.
