Impact of microgravity on retinal neuroimmune responses and visual dysfunction in rats

Abstract

Aim: To analyze visual dysfunction in rats under simulated weightlessness (SW) by examining trans-laminar cribrosa pressure difference (TLCPD) and neuroimmune responses.

Methods: The 72 male Sprague-Dawley rats were randomly assigned into two groups (ground control and hindlimb unloading-simulated microgravity) using stratified randomization, with each group further subdivided into three exposure durations: SW 2-week (SW-2W), 4-week (SW-4W), and 8-week (SW-8W), n=12 per subgroup. At the designated time points for each group, intraocular pressure (IOP) and intracranial pressure (ICP) were measured, and the trans-laminar cribrosa pressure difference (TLCPD) was calculated. Additionally, optomotor response (OMR), electroretinography (ERG), and optical coherence tomography (OCT) were performed. The number of retinal ganglion cells (RGCs) was quantified via immunofluorescence, the activation of astrocytes and microglial cells was determined, and Sholl analysis was conducted to assess the function and morphology of microglial cells. Data were analyzed with SPSS and GraphPad Prism (P<0.05).

Results: Under prolonged simulated microgravity, rats exhibited a progressive increase in both IOP and ICP, with the most pronounced rise observed at 8wk. Concurrently, the TLCPD shifted from a negative value in controls to a positive value. These pressure alterations were associated with retinal dysfunction, as evidenced by significant reductions in ERG b-wave and photopic negative response (PhNR) amplitudes. OCT and histological analyses revealed subtle photoreceptor layer damage: while the inner nuclear layer (INL) thickness remained relatively unchanged, the outer nuclear layer (ONL) thinned significantly, and the nerve fiber layer-ganglion cell layer complex thickness (NFL-GCL) complex initially thickened before later thinning. Immunofluorescence further demonstrated marked neuroimmune activation, with astrocytes transitioning from having large cell bodies with small, elongated, sparse processes to a phenotype characterized by compact, enlarged nuclei and aggregated processes, alongside notable RGC loss.

Conclusion: Based on the results from the simulated microgravity rat model, microgravity-induced changes in dual-chamber pressure, and neuroimmune responses in the retina may play a key role in visual dysfunction. Specifically, the activation of retinal neuroimmune cells (astrocytes and microglial cells) induced by mechanical stress appears to be central to retinal and optic nerve damage.

Keywords: astrocyte activation; microglia; microgravity; neuroimmune; simulated weightlessness.

Figure 1. Overall study timeline of SW in rats

The 72 male Sprague-Dawley rats were randomly assigned to two groups (ground control and hindlimb unloading-simulated microgravity) using stratified randomization, with each group further subdivided into three exposure durations: 2-week (SW-2W, n=12), 4-week (SW-4W, n=12), and 8-week (SW-8W, n=12) subgroups. At the designated time points, each group underwent assessments, including IOP measurements, OCT, ERG, and OMR testing. At the final stage, ICP was measured through surgical procedures. Following the completion of all assessments, the rats were euthanized with a lethal dose of sodium pentobarbital. OMR: Optomotor response; ERG: Electroretinogram; ICP: Intracranial pressure; OCT: Optical coherence tomography; SW: Simulated weightlessness; IOP: Intraocular pressure.

Figure 2. IOP, ICP, and TLCPD in SW rats

A: IOP values in rats (n=12); B: ICP values in rats (n=8); C: Line graph depicting changes in IOP and ICP pressure. D: TLCPD results in SW rats (n=8). E: Line graph showing TLCPD results in both SW and control rats (n=8). ^aP<0.05 between the SW group and the control group at the same time point. ^bP<0.01, ^cP<0.001, ^dP<0.0001. IOP: Intraocular pressure; ICP: Intracranial pressure; SW: Simulated weightlessness; TLCPD: Trans-lamina cribrosa pressure difference; CTRL: Control.

Figure 3. Visual function assessment in SW rats

A: Schematic representation of the a-wave, b-wave, and PhNR waveforms in ERG response; B: OMR test conducted on rats in a darkroom; C: Spatial frequency contrast sensitivity (C/D) derived from OMR testing in SW rats; D: a-wave in the ERG of SW rats; E: b-wave in the ERG of SW rats; F: PhNR wave in the ERG of SW rats, ^eP<0.05 (n=12), ^aP<0.05 between the SW group and the control group at the same time point. ERG: Electroretinogram; OMR: Optomotor response; SW: Simulated weightlessness; PhNR: Photopic negative response; CTRL: Control.

Figure 4. Retinal thickness measurements in SW rats using OCT

A: Full retinal thickness (µm); B: NFL-GCL thickness (µm); C: INL thickness (µm); D: ONL thickness (µm); E: OCT image of the retina in rats. ^aP<0.05 between the SW group and the control group at the same time point. ^eP<0.05, ^bP<0.01, ^cP<0.001, ^dP<0.0001 (n=12). OCT: Optical coherence tomography; INL: Inner nuclear layer; NFL-GCL: Nerve fiber layer and ganglion cell layer complex; ONL: Outer nuclear layer; SW: Simulated weightlessness; CTRL: Control.

Figure 5. In vivo fundus imaging and H&E-stained retinal sections of rats under SW

A–D: Fundus photographs from each experimental group (n=12 eyes per group/time point); E–H: Representative H&E-stained retinal sections from all groups; I–L: Regions outlined in black indicate the areas where retinal structures were analyzed (n=3 eyes per group/time point). H&E: Hematoxylin-eosin; RCG: Retinal ganglion cell layer; IPL: Inner plexiform layer; INL: Inner nuclear layer; ONL: Outer nuclear layer; SW: Simulated weightlessness.

Figure 6. Immunofluorescence analysis of retinal changes in rats under SW

A–D: Representative retinal flatmount images demonstrating RBPMS immunostaining (gray) for RGCs at 2, 4, and 8wk post-modeling, along with quantitative analysis of RGC numbers (n=11 eyes per group/time point); E–H: Representative retinal flatmount images illustrating GFAP immunostaining (red) at 2, 4, and 8wk post-modeling (n=3 eyes per group/time point); I-L: Representative images of Iba-1 immunostaining (green) in retinal flatmounts at 2, 4, and 8wk post-modeling, with quantification of microglia density (n=6 eyes per group/time point). RGC: Retinal ganglion cells; SW: Simulated weightlessness; RBPMS: RNA-binding protein with multiple splicing; GFAP: Glial fibrillary acidic protein.

Figure 7. Activation and morphological changes of retinal microglia in rats under SW

A: Representative images of Iba-1 staining were converted to binary format for Sholl analysis, and the resulting Sholl profiles for rats exposed to SW at 2, 4, and 8wk are presented (n=6 eyes per group/time point); B–D: Skeleton analysis was used to quantify the number of intersections across groups; E: Maximum branch length; F: Average branch length; G: Total branch number were quantified. ^aP<0.05 between the SW group and the control group at the same time point. SW: Simulated weightlessness; CTRL: Control.

Figure 8. Schematic of TLCPD

TLCPD is defined as the difference between IOP and ICP. Under normal conditions, IOP exceeds ICP, resulting in a pressure gradient directed toward the posterior of the eyeball. Under SW, the pressure gradient is reversed, pointing toward the anterior of the eyeball. In special cases, such as glaucoma—where IOP is substantially higher than ICP—the resulting large pressure gradient is directed toward the posterior of the eyeball. TLCPD: Trans-lamina cribrosa pressure difference; IOP: Intraocular pressure; ICP: Intracranial pressure; SW: Simulated weightlessness.

Figure 9. Reversed TLCPD causes mechanical stress on the lamina cribrosa region of the retina, leading to damage of retinal nerve cells

A: Under simulated microgravity, when ICP exceeds IOP, a pressure gradient directed anteriorly is generated. In this pathological state, the reversed pressure difference is considered in our study to cause damage to retinal photoreceptor cells, resulting in impaired visual function. B: Activation of two types of neuroimmune cells in our study—astrocytes and microglial cells—transitioning from a resting state to an activated state. C: Excessive activation of neuroimmune cells may ultimately damage retinal cells, as evidenced by findings in ERG, OMR, histopathological sections, and immunofluorescence staining. TLCPD: Trans-lamina cribrosa pressure difference; IOP: Intraocular pressure; ICP: Intracranial pressure; SW: Simulated microgravity; ERG: Electroretinography; OMR: Optomotor response; CTRL: Control.