Are Temporal Differences in GDNF and NOS Isoform Induction Contributors to Neurodegeneration? A Fluorescence Microscopy-Based Study

Abstract

Background: Specific factors in Parkinson's disease have become targets as to their protective and degenerative effects. We have demonstrated that cytokines and PD-CSF detrimentally affect microglia and astrocyte growth. While glial cell-derived neurotrophic factor (GDNF) has been recognized as a possible neuron-rescue agent, nitric oxide synthase (NOS) has been implicated in neurodegenerative processes.

Objective: To demonstrate that glial cell activation, cytokine production, and NOS induction, play an intimate role in the loss of dopaminergic signaling, via mechanisms that are a result of inflammation and inflammatory stimuli.

Methods: Study animals were sacrificed following endotoxin treatment and tissue sections were harvested and probed for GDNF and NOS isomers by fluorescence deconvolution microscopy. Fluorescence was mapped and quantified for each probe.

Results: An immune cell influx into 'vulnerable' areas of the brain was seen, and three NOS isomers, inducible (iNOS), neuronal (nNOS) and endothelial (eNOS), were synthesized in the brains, a finding which suggests that each isomer has a role in neurodegeneration. eNOS was found associated with blood vessels, while iNOS was associated with glial and matrix cells and nNOS was located with both glia and neurons. Following endotoxin treatment, serum levels of nitric oxide were higher at 6-8 hours, while tissue levels of NOS were elevated for much longer. Thus, induction of NOS occurred earlier than the induction of GDNF.

Conclusion: Our findings suggest that the protective abilities of GDNF to combat neural destruction are not available rapidly enough, and do not remain at sufficiently high levels long enough to assert its protective effects. (250).

Keywords: Endotoxin; Fluorescence microscopy; Glial derived neurotrophic factor; Neurodegeneration; Nitric oxide.

Fig. (1)

Circulating nitric oxide levels after LPS treatment peak at 6-12 hours but remain above basal levels even after 48 hours.

Fig. (2)

Shows data that attests to a robust inflammatory response. The graph on the left (Panel A - Cell Types) details the increasing number of lymphocytes in the olfactory bulb >6 hours, while cytokines also increase in concentration (Panel A - Proteins), particularly TNFα and interleukin-6. The GDNF concentration also reveals a gradual increase, up to at least 12 hours. The fluorescent image reveals a dense cuff of lymphocytes (yellow) around an arteriole in the olfactory bulb (light green). Nuclei are blue and the dark green indicates the matrix (Magnification x 600) (Panel B).

Fig. (3)

LPS treatment resulted in a substantial, sustained increase in inducible nitric oxide synthase (iNOS) for at least 12 hours Fig. (3) shows images from sections of olfactory bulb at 2 and 8 hours post treatment), with iNOS (Red) clearly visible concentrated along the nuclei (blue) of sensory cell tracts. The connective tissue matrix is shown in green (Magnification x 900). Cortical tissue was used as “control” as our initial experiments concentrated on the olfactory bulb as loss of olfaction is an early indicator of PD, but when comparing cortex and olfactory bulb there was a substantial increase in iNOS in both areas, attesting to a major and prolonged inflammatory response, but these images not shown.

Fig. (4)

eNOS (orange/red) was concentrated around vessels, at time 0, 2 and 8 hours after LPS treatment, with capillaries clustered around an arteriole in panel A. An arteriole (blue arrow) and a venule (yellow arrow) are shown in panel B, and two arterioles are evident in panel C. The images show a ‘separation’ of the eNOS from a medial location to an adventitia area, 2 hours after treatment. This change was not seen at time zero, or 8 hours after treatment (magnification x 600).

Fig. (5)

To have a structural view of eNOS (red) with vessels, we made rotatable, 3D models Fig. (5), showing eNOS around a venule (panel A) and an arteriole (panel B) in a section of olfactory bulb, demonstrating specificity of this NOS isoform to endothelial cells. Smaller vessels can also be seen coursing throughout the tissue. Connective tissue staining has been removed for clarity (Magnification x 1200).

Fig. (6)

is a compilation of images to demonstrate both comparative and methodological properties of image renditions. Panels A, B, C show combinations of nNOS (red), connective tissue (green) and nuclei (blue) as a complete-field stack of all three probes (A), then with the connective tissue removed (B), and finally with part of the image ‘wire-framed’ (Mag x 900) to visualize juxta-positioning of nNOS to assess specificity.