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Methylnitrosourea MNU-induced Retinal Degeneration and Regeneration in the Zebrafish: Histological and Functional Characteristics
Published on: October 20, 2014
Dynamic changes in microglial and macrophage characteristics during degeneration and regeneration of the zebrafish
Diana M Mitchell1, Anna G Lovel2, Deborah L Stenkamp2
1Department of Biological Sciences, University of Idaho, 875 Perimeter Drive, MS 3051, Moscow, ID, 83844-3051, USA. dmitchell@uidaho.edu.
Background:
In contrast to mammals, zebrafish have the capacity to regenerate retinal neurons following a variety of injuries. Two types of glial cells, Müller glia (MG) and microglia, are known to exist in the zebrafish retina. Recent work has shown that MG give rise to regenerated retinal neurons, but the role of resident microglia, and the innate immune system more generally, during retinal regeneration is not well defined. Specifically, characteristics of the immune system and microglia following substantial neuron death and a successful regenerative response have not been documented.
Methods:
The neurotoxin ouabain was used to induce a substantial retinal lesion of the inner retina in zebrafish. This lesion results in a regenerative response that largely restores retinal architecture, neuronal morphologies, and connectivities, as well as recovery of visual function. We analyzed cryosections from damaged eyes following immunofluorescence and H&E staining to characterize the initial immune response to the lesion. Whole retinas were analyzed by confocal microscopy to characterize microglia morphology and distribution. Statistical analysis was performed using a two-tailed Student's t test comparing damaged to control samples.
Results:
We find evidence of early leukocyte infiltration to the retina in response to ouabain injection followed by a period of immune cell proliferation that likely includes both resident microglia and substantial numbers of proliferating, extra-retinally derived macrophages, leading to rapid accumulation upon retinal damage. Following immune cell proliferation, Müller glia re-enter the cell cycle. In retinas that have regenerated the layers lost to the initial injury (histologically regenerated), microglia retain morphological features of activation, suggesting ongoing functions that are likely essential to restoration of retinal function.
Conclusions:
Collectively, these results indicate that microglia and the immune system are dynamic during a successful regenerative response in the retina. This study provides an important framework to probe inflammation in the initiation of, and functional roles of microglia during retinal regeneration.
Insights
Zebrafish immune cells, including microglia, dynamically respond to retinal injury, supporting neuron regeneration. Activated microglia play essential roles in restoring retinal structure and function after damage.
Area of Science:
- Ophthalmology
- Neuroscience
- Immunology
Background:
- Zebrafish exhibit remarkable retinal regeneration, unlike mammals.
- Müller glia (MG) are known neuron progenitors, but the immune system's role, particularly microglia, in zebrafish retinal regeneration is unclear.
- The characteristics of immune cells during successful retinal regeneration after significant neuron loss remain undocumented.
Purpose of the Study:
- To investigate the role and characteristics of microglia and the innate immune system during zebrafish retinal regeneration.
- To document immune cell dynamics following induced retinal damage and subsequent regeneration.
Main Methods:
- Ouabain-induced inner retinal lesions in zebrafish to model injury and regeneration.
- Immunofluorescence and H&E staining of cryosections to analyze immune response.
- Confocal microscopy of whole retinas to assess microglia morphology and distribution.
- Statistical analysis using two-tailed Student's t tests.
Main Results:
- Early leukocyte infiltration and subsequent immune cell proliferation, including resident microglia and infiltrating macrophages, were observed.
- Müller glia re-entered the cell cycle post-immune cell proliferation.
- Activated microglia morphology persisted in histologically regenerated retinas, suggesting ongoing functional importance.
Conclusions:
- Microglia and the immune system exhibit dynamic behavior during successful retinal regeneration in zebrafish.
- This study establishes a foundation for further research into inflammation's role in initiating and the functional significance of microglia in retinal regeneration.
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