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 probeResults: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).

Changes in Vascular and Immune Cell Content of an LPS Treated Rat Olfactory Bulb PD Model, Measured by Fluorescence Deconvolution Microscopy

“…Increases in immune cell numbers were noted as shown in low magnification images (Figure 2), visualizing T cells, B cells and macrophage numbers over the first 6 h. These findings suggest the peak response time of the acute phase of the inflammation, when enough GDNF is present to overcome at least some of the endotoxin assault, cytokine-initiated cell death and glial cell activation [5]. Of note, is the reduction in the number of macrophages and neutrophils after an initial increase, while B-cell numbers remain high at 6 h and T-cell content continues to increase.…”

“…Deconvolution fluorescence microscopy 3D image reconstructions of NOS isoforms, actin and nuclei were generated [5,21,29] and images were saved as TIFF files. Comparative statistical analyses (one way ANOVA) of fluorescence densities (pixel numbers) were made using Corel (Ottawa, Ontario, Canada) and SigmaStat software (SPSS, Chicago, IL, USA).…”

“…Many factors play a role in chronic neurodegeneration which leads to Parkinson's disease (PD), including inflammation [1], cytokine syntheses and release [1], protein recycling-ubiquitination problems [2], glial cell activation [3], loss of astrocyte support [4], neurotrophic factors [2], nitric oxide synthase [5] and mitochondrial dysfunction [2]. However, recent reports have shown that circulating cells [6] are also involved in disease progression, which coupled with vascular damage [7], points to both peripheral and innate factors and cell involvement in the ensuing demise of neurons.…”

“…Previous work in our lab has demonstrated the 'toxicity' of PD-CSF [20] including, the presence of non-resident cells and cytokine production in the olfactory bulb of an LPS rat model [21], specific localizations of PD-associated proteins and cytokines in LPS treated brain [22] and the induction of neurotrophins and nitric oxide [5]. We therefore continued our studies to further understand the progression of this chronic, debilitating disease, by correlating cytokine, protein and neurotrophic factor concentrations and localizations, together with immune cell numbers and a loss of vascular integrity.…”

“…Using fluorescent tags and deconvolution imaging we examined vascular content, vessel integrity and vessel types in images of LPS treated rat olfactory bulbs and concluded that, by and large, vessels are overall intact but with 'suspect' areas. These findings might indicate that even though protective GDNF is produced and nitric oxide is released to maintain vascular tone, subsequent inflammatory episodes are increasingly challenging due to the first assault making repair and rescue difficult due to a loss of BBB control, continuing increases in cytokine levels and reductions in normal cellular abilities [5].…”