The Role of HDAC3 in Retinal Ischemia-Reperfusion Injury

Background

Retinal ischemia-induced retinal diseases are significant characteristics of common visual impairments such as diabetic retinopathy (DR) and central retinal artery and vein occlusion. The therapeutic effects for these ischemic retinal diseases are generally ineffective, and the design of new therapies urgently requires an in-depth understanding of the related disease mechanisms. Histone deacetylases (HDACs) are essential enzymes that regulate gene expression and protein functions. Preclinical studies have shown that non-specific HDAC inhibitors can reduce retinal injury. Among all HDACs, histone deacetylase 3 (HDAC3) is a class I histone deacetylase subtype and plays a core role in the inflammatory response of macrophages. Our previous studies found that HDAC3 in myeloid cells was upregulated in a mouse retinal ischemia-reperfusion (IR) injury model. However, it is currently unclear whether this cellular event essentially contributes to retinal IR injury.

Study Source

This research was conducted by Rami A. Shahror, Esraa Shosha, Carol Morris, Melissa Wild, Shengyu Mu, Gabor Csanyi, Marjan Boerma, Nancy J. Rusch, and Abdelrahman Y. Fouda (*corresponding author) from University of Arkansas for Medical Sciences, Augusta University, and Cairo University. The article was published in the Journal of Neuroinflammation, Volume 21, 2024.

Research Process

Study Subjects and Process

The study subjects included myeloid cell-specific HDAC3 knockout mice (M-HDAC3 KO) and control group mice. These mice were used for retinal IR injury experiments and observed through a series of methods. These methods included optical coherence tomography (OCT), electroretinography (ERG), Western blot, and immunofluorescence labeling to specifically ascertain the impact of HDAC3 on retinal neurovascular injury.

The specific experimental steps are as follows:

1. Establishment of Mouse Retinal Ischemia-Reperfusion Model: After anesthetizing the mice, retinal ischemia was induced by increasing intraocular pressure to 110 mmHg and maintaining it for 60 minutes, followed by removing the needle to achieve reperfusion.

2. Processing of Mouse Samples: Samples were taken at different time points (2 days, 7 days, 14 days) for related detection, such as observing vascular permeability, retinal thickness, neural degeneration, and visual function.

3. Drug Treatment and Groups: Specific HDAC3 inhibitors (RGFP966) were administered to wild-type mice, and the retinal IR injury was observed along with its protective effect.

Experimental Details

1. Optical Coherence Tomography (OCT): After 7 days of retinal IR, retinal thickness was scanned and obtained from live mice using OCT to evaluate its integrity.

2. Electroretinography (ERG): Deep visual responses were recorded, quantifying wave amplitude and inner retinal function under dim light stimulation.

3. Vascular Permeability Detection: The integrity of retinal blood vessels was analyzed by Western blot, fluorescein angiography, and Evans blue staining.

4. Myeloid Cell Response Detection: Flow cytometry was used to quantitatively analyze the myeloid cell response in the retina, assessing the cell populations marked by CD45 and CD11b.

5. Function of Clearing Apoptotic Cells by Myeloid Cells: Confocal microscopy and in vitro phagocytosis experiments were used to detect differences in the clearance of dead cells by myeloid cells with and without HDAC3.

Major Research Results

1. Expression of HDAC3 in Mouse and Human Retina: HDAC3 was upregulated in myeloid cells after retinal IR injury in mice, and HDAC3 also showed demethylated enzyme expression in retinal samples from patients with diabetic retinopathy.

2. Protective Effect of HDAC3 Knockout on Neurodegeneration Post-Retinal IR: HDAC3 knockout mice showed preserved neuronal cells, maintained vascular integrity, and protected retinal thickness.

3. Improved Visual Function: ERG testing showed that mice lacking HDAC3 had significantly better visual function after retinal IR injury compared to the control group.

4. Impact on Myeloid Cell Proliferation and Infiltration: HDAC3 knockout mice showed lower myeloid cell proliferation and infiltration.

5. Enhanced Cell Clearance Effect by HDAC3 Knockout: Myeloid cells lacking HDAC3 exhibited higher phagocytic activity of apoptotic cells.

6. Effect of Pharmacological Inhibition of HDAC3: Administering the HDAC3 inhibitor RGFP966 to wild-type mice resulted in significant neuroprotection and retinal integrity preservation, similar to the effects observed with HDAC3 gene knockout.

Research Conclusion

This study provides the first direct evidence that HDAC3 promotes retinal ischemia-reperfusion injury by inhibiting phagocytosis of dead cells in myeloid cells, and that deleting HDAC3 can enhance the myeloid cells’ repair mechanism of clearing dead cells, contributing to injury mitigation. This offers a new therapeutic strategy targeting HDAC3 for potential new treatments to protect the integrity and functional recovery of the retina after ischemic injury.

Research Highlights

1. Unveiling a New Mechanism: For the first time, it reveals the inhibitory effect of HDAC3 on myeloid cell phagocytosis of dead cells and identifies HDAC3 as a critical molecule promoting retinal IR injury.
2. Diverse Technical Methods: Multiple modern techniques were used, including OCT, ERG, Western blot, and flow cytometry, providing robust results.
3. Potential Application Value: The HDAC3 inhibitor RGFP966 exhibited similar protective effects to HDAC3 gene knockout in mice, indicating its potential clinical application.

Significance and Value

This research confirms HDAC3 as an essential negative regulator in retinal ischemia-reperfusion injury, revealing its mechanism of inhibiting myeloid cell clearance of apoptotic cells and providing a new therapeutic target for treating ischemic retinal diseases. Additionally, the application of RGFP966 demonstrates the therapeutic potential of HDAC3 inhibitors. This research also lays the foundation for in-depth studies of ischemic retinal diseases, potentially driving the development of novel treatments to improve clinical outcomes.

The entire study provides new perspectives and potential strategies for the treatment of retinal ischemia-reperfusion injury, bearing significant scientific and clinical implications.