Pharmacological Activation and Transgenic Overexpression of SIRT1 Attenuate Traumatic Optic Neuropathy Induced by Blunt Head Impact

Abstract

Purpose: Resveratrol (RSV) is a nutraceutical compound known for its therapeutic potential in neurodegenerative and metabolic diseases. RSV promotes survival signals in retinal ganglion cells (RGCs) through activation of SIRT1, an NAD⁺-dependent deacetylase. RSV and SIRT1 reduce RGC loss induced by direct optic nerve injury, but effects in indirect models of traumatic optic neuropathy remain unknown and are examined in this study.

Methods: An electromagnetic stereotaxic impactor device was used to impart five traumatic skull impacts with an inter-concussion interval of 48 hours to wild type (WT) and SIRT1 knock in (KI) C57BL/6J mice overexpressing the SIRT1 gene. A cohort of WT mice also received intranasal administration of RSV (16 mg/kg) throughout the experimental period. Loss of righting reflex (RR), optokinetic response (OKR) scores, and immunolabeled RGC count are determined to assess optic neuropathy in this model of traumatic brain injury (TBI).

Results: TBI significantly decreases RGC survival and decreases OKR scores compared with control uninjured mice. Either RSV administration in WT mice, or SIRT1 overexpression in SIRT1 KI mice, significantly increases RGC survival and improves OKR scores. RR time increases after the first few impacts in all groups of mice subjected to TBI, demonstrating that RSV and SIRT1 overexpression are able to attenuate optic neuropathy following similar degrees of TBI.

Conclusions: Intranasal RSV is effective in preserving visual function in WT mice following TBI. Constitutive overexpression of SIRT1 recapitulates the neuroprotective effect of RSV.

Translational Relevance: Results support future exploration of RSV as a potential therapy for indirect traumatic optic neuropathy.

Introduction

Traumatic optic neuropathy (TON) is a condition in which acute injury to the optic nerve from direct or indirect trauma results in permanent blindness.¹ Blunt trauma to the skull which occurs as a result of sports-related injuries, falls, assaults, or motor vehicle accidents, imparts deformative stress to the intracanalicular segment and/or intracranial part of the optic nerve which is susceptible to traumatic shock injury.^2,3 Shearing of retinal ganglion cell (RGC) axons ensues which leads to a dramatic neuronal loss and irreversible vision deterioration with little or no possibility of recovery. Currently, there is no accepted effective treatment for TON. Previous studies reported potential effects of megadoses of steroid-based treatments for TON.^4,5 However, those, and trials examining other potential therapies, were small, imperfectly executed, lacked adequate randomization, and rigorous statistical analyses, and none of them identified convincing positive functional visual outcomes with pharmacological and/or surgical interventions.^6,7 Causative factors that have been linked to RGC death and subsequent vision loss include, but are not limited to, optic nerve swelling, inflammation, increased oxidative stress and intracellular calcium, and disruption of the blood barrier.^8,9 Large amounts of reactive oxygen species (ROS) produced as a result of neuronal injury destroy the structure and function of mitochondria, causing oxidative stress condition-induced cell death.¹⁰ Targeting the deleterious effects of these secondary injury processes may help mitigate RGC vulnerability to damages caused by external physical insults. 

Histone deacetylases (HDACs) are a group of enzymes that catalyze the removal of acetyl groups from ε-N-acetyl lysine residues of histones and non-histone proteins.¹¹ The 18 different HDACs that have been identified in humans are grouped into two families (“classical” and “sirtuins” or silent information regulator [SIRTs]). SIRT1 is a member of the sirtuin family and a ubiquitously expressed NAD⁺-dependent deacetylase regulating the activity and expression of histones and non-histone transcription factors, including Nrf2, NF-kB, PDX-, forkhead box class O, and peroxisome proliferator activated receptors γ Coactivator 1α (PGC-1α).^12,13 As such, SIRT1 plays a pivotal role in a variety of biological and metabolic processes, including inflammation, oxidation, and energy metabolism.^14,15 An increasing number of studies have shown that expression and activity of SIRT1 reduced neurodegenerative diseases of the central nervous system (CNS), including Alzheimer's and Parkinson's diseases.^16,17 Yang et al. have shown that pharmacological activation of SIRT1 by resveratrol (RSV), a stilbene natural polyphenol and one of the natural agonists of SIRT1, is able to cross the blood brain barrier, reduce brain injury, and attenuate brain tissue apoptosis in neonatal pups subjected to hyperoxic injury.¹⁸ Studies from our own group have shown that a pharmaceutical formulation of RSV prevented neuronal damage and associated long-term neurologic dysfunction in a relapsing-remitting mouse model of experimental autoimmune encephalomyelitis (EAE),^19,20 and a recent study demonstrated similar effects of intranasal RSV in a chronic EAE mouse model.²¹ Similarly, Yu et al. have shown that pharmacological activation of SIRT1 improved post-traumatic brain injury (TBI) cognitive function through activation of the PGC-1α pathway, a key factor in ameliorating mitochondrial function and reducing ROS production.²² This protective effect of SIRT1 on neurological function suggests its potential beneficial effect in preventing/ ameliorating TON-associated damage to RGCs and indeed we found that RSV at least partially delays RGC loss induced by direct optic nerve crush injury.²³ Studies from our group have further shown that traumatic skull impacts to C57BL/6J mice induced bilateral RGC loss and altered visual function when compared with control non-impacted mice.²⁴ This model provides the opportunity to explore potential neuroprotective effects of SIRT1 activation or overexpression in TBI-induced TON. 

Materials and Methods

Mice

C57BL/6J wild type (WT) and SIRT1 knock in (KI) mice (stock #013080) purchased from The Jackson Laboratory (Bar Harbor, ME, USA) were housed and bred at the University of Pennsylvania animal facility. The mice were raised in a 12-hour light/dark cycle at an ambient temperature of 22°C until approximately 3 to 4 months of age prior to induction of TBI. SIRT1 KI mice contained SIRT1 cDNA knocked into the beta-actin locus.²⁵ Animal breeding and treatment conformed to Institutional Animal Care and Use Committee guidelines, the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research, and the Association for Assessment and Accreditation of Laboratory Animal Care International. 

Traumatic Brain Injury

The TBI procedure used was implemented as described previously.²⁴ Briefly, mice had the fur on their heads shaved and once anesthetized with isoflurane, were placed on stereotaxic equipment (Impact One Stereotaxic Impactor; Leica Biosystems, Buffalo Grove, IL, USA). The impact device was equipped with a 5 mm cylindrical flat head hammer and struck at a velocity of 5 mm/s with a strike depth of 1.0 mm. Five impacts were delivered with the anterior edge of the impactor hitting the bregma of the skull with a 48-hour interval between impacts. Control mice received anesthesia of similar duration to the experimental mice but did not receive impact. 

Righting Reflex

Delay of the righting reflex (RR) was measured immediately post-impact. It is a mesencephalic reflex that recovers function from unconsciousness due to brain injury or anesthesia earlier than thalamocortical activation.²⁶ This behavioral assessment can function as an indicator for injury severity. RR is assessed by placing the mouse in the supine position immediately after impact and recording the time from being placed in such a position to the time the animal shows the first signs of righting themselves. The mice were placed in a small container under a heat lamp. 

Optokinetic Response

Visual function was measured by optokinetic response (OKR) using OptoMetry software (Cerebral Mechanics, Inc., Lethbride, AB, Canada). The OKR scores were recorded prior to and after TBI treatments, as previously described.²⁷ OKR was measured prior to the first impact at day –6. The measurements were repeated on day 10 and again once a week until euthanization on day 44. Time of euthanization was selected based on prior studies demonstrating maximal visual dysfunction and RGC loss that occur by 6 weeks after initial impact.²⁴ 

RGC Labeling and Quantification

Mouse eyes were harvested at day 44 and placed in 4% paraformaldehyde (PFA) for 1 hour at room temperature. The retinas were then dissected and processed, as previously described.²⁸ After 3 washes with phosphate-buffered saline (PBS), the retinas were permeabilized in 0.5% Triton X-100 in PBS by freezing for 15 minutes at –70°C. Retinas were subsequently incubated overnight at 4°C in a humidified chamber with rabbit anti-Brn3a antibody (Synaptic Systems, Goettingen, Germany) diluted 1:1000 in a blocking buffer containing 2% bovine serum albumin and 2% Triton X-100. The tissues were washed 3 times in PBS and then incubated for 1 hour at room temperature with Alexa Fluor 488 anti-rabbit secondary antibody (Invitrogen, Rockford, IL, USA) diluted 1:1000 in blocking buffer. After staining, photographs were taken by a masked investigator using a fluorescence microscope and Nikon NIS-Elements Imaging Software at times 40 magnification in 12 standard fields: 1/6, 3/6, and 5/6 of the retinal radius from the center in each quadrant, covering a total area of 0.431 mm²/retina. Masked investigators then counted the number of Brn3a+ RGCs using ImageJ software. RGC counts were averaged across the 12 representative fields, or averaged across 4 fields each from the central (1/6 radius), mid-peripheral (3/6), and peripheral (5/6) retinal regions, as indicated in results/figure legends. 

RSV Treatment

Experimental cohorts of WT mice were treated intranasally with 16 mg/kg RSV suspended in 20 µL PBS, or PBS alone for control cohorts. The RSV dose was selected based on efficacy of intranasal RSV in preventing RGC loss during optic neuritis in EAE mice.²¹ This procedure was carried out daily starting on the day of TBI until euthanization. 

Axon Staining and Quantification

Isolated optic nerves were fixed in 4% PFA for 1 hour at room temperature, then washed, processed, imbedded in paraffin, and cut in 5 µm thick longitudinal sections. Sections of the optic nerve were deparaffinized, rehydrated, and then permeabilized with 0.5% tween-20 in PBS. Sections were incubated in rabbit anti-neurofilament antibody 1:100 (Abcam ab8135) at 4°C overnight, washed, and then incubated for 1 hour at room temperature with Alexa Fluor 488 anti-rabbit secondary antibody (Invitrogen, Rockford, IL, USA) diluted 1:1000 in blocking buffer. After staining, grayscale photographs of the center of the optic nerve were taken by a masked investigator using a fluorescence microscope and Nikon NIS-Elements Imaging Software at 20 times magnification. Staining was quantified by calculating the mean fluorescence value (i.e. the mean gray value of grayscale images) using Image J software (nih.gov), minus the mean fluorescence value of a serial optic nerve section stained with secondary antibody only (background staining). The right optic nerve of each mouse was used for quantitative comparisons between treatment groups. 

Statistics

All data are represented as mean ± SEM. Differences between treatment groups with respect to RR, OKR responses, and RGC quantification were determined using a 1-way ANOVA followed by Tukey's honest significant difference test using statistical software (GraphPad Prism 5.0; GraphPad Software, Inc., La Jolla, CA, USA). Differences were considered statistically significant at P < 0.05. 

Results

Effects of RSV on Visual Function in Mice With TBI

Figure 1 shows a schematic representation of the stereotaxic impactor device used to induce TON in C57BL/6J mice. With its 5 mm cylindrical flat head hammer, the impactor strikes the mouse head at a velocity of 5 mm/s with a strike depth of 1.0 mm. The hammer was driven by a pneumatic piston, making the force of the impacts reproducible from mouse to mouse. We used this device to examine the therapeutic properties of RSV on visual function and RGC survival following TBI. For this purpose, isoflurane-anesthetized mice were organized into three groups: (1) a control uninjured untreated group that was anesthetized and placed on the stereotaxic device but no impacts were delivered; (2) an experimental TBI group sham treated with daily intranasal injection of PBS; and (3) an experimental TBI group treated with daily intranasal RSV (16 mg/kg). Mice in both TBI groups each received a single impact targeted to the bregma of the skull on days 0, 2, 4, 6, and 8. To confirm that brain injury was successfully induced, and determine whether RSV administration affects cognitive behavior and balance in TBI mice, we assessed the RR by placing the mouse in the supine position immediately after impact (or sham impact for control mice) and recording the time required to upright itself with all four feet on the ground. As shown in Figure 1B, the control mouse group shows the same RR time throughout the time course of the experiment. In contrast, both the TBI group receiving PBS and the TBI group receiving RSV showed an increase of RR time that was highest following the initial two impacts, then steadily improved, but overall was significantly higher than controls across all five impacts, as analyzed by ANOVA of repeated measures. The pattern of RR times was similar between the TBI groups, with no statistically significant change of RR time between RSV and PBS treated TBI groups. 

Figure 1.

 

Effects of TBI and RSV treatment on RR time. (A) Schematic representation of the stereotaxic impactor device used to induce TBI. (B) Effects of RSV on the RR time in WT mice. RR time was determined in isoflurane-anesthetized mice of the control (no TBI, n = 3) mice, TBI (n = 6), and TBI + RSV (n = 3) groups. Mice of the latter 2 groups received, each, a single impact on days 0, 2, 4, 6, and 8 on their heads. These two groups also received intranasal administration of PBS and RSV (16 mg/kg daily), respectively, throughout the duration of the experiment. RR time was defined as the time from placement in the supine position until the animals turned all four paws and stood upright. Data are means ± SEM. RR times were compared by repeated measures ANOVA (**P < 0.001).

We further evaluated visual function in all three groups of mice by determining OKRs. Control animals showed unaltered visual function reflected by consistent OKR scores averaged between both eyes throughout the 6-week time course of the experiment (Fig. 2A). In contrast, the TBI group treated with PBS showed a progressive decline of OKR scores over the initial 3 weeks following the first impact, and plateaued thereafter. However, the group of TBI mice treated with RSV showed OKR scores that were comparable to those of control, uninjured mice. Across 6 weeks, OKR scores of RSV-treated TBI mice were significantly higher than those of PBS-treated TBI mice. Because the impact on the mouse head was equidistant from both eyes, bilateral vison loss was anticipated. Concordantly, the changes of OKR scores of either the right or left eyes were analyzed separately. As shown in Figures 2B and 2C, OKR scores in each eye were similar to those measured for both eyes, suggesting that RSV treatment of TBI mice attenuated visual function alterations to a similar degree in right and left eyes. 

Figure 2.

 

Intranasal administration of RSV attenuates visual function loss following TBI. (A) OKR scores averaged between left and right eyes show a significant (***P < 0.001) decrease over a 6-week time course in TBI mice (n = 6) compared with control (no TBI, n = 3) mice. Daily intranasal RSV administration significantly attenuated vision loss in TBI + RSV mice (n = 3, ***P < 0.001) compared with TBI mice. There was no significant difference in visual function between TBI + RSV and control no TBI mice. (B) OKR scores of the left eye show a significant (**P < 0.01) progressive decrease in TBI mice compared with control mice. RSV significantly attenuated vision loss in TBI + RSV mice (***P < 0.001) compared with TBI mice. There was no significant difference in visual function between TBI + RSV and control no TBI mice. (C) OKR scores of the right eye show a significant (**P < 0.01) progressive decrease in OKR scores in TBI mice as compared with control mice. RSV significantly attenuated vision loss in TBI + RSV mice (**P < 0.01) compared with TBI mice. There was no significant difference in visual function between TBI + RSV and control mice. OKR scores of all panels were compared by repeated measures ANOVA. Data from one of three representative experiments are shown.

Effects of RSV Treatment on RGC Survival

We determined the RGC number in retinal flat mounts of the uninjured and injured PBS- and RSV-treated groups by counting Brn3a-immunolabeled RGCs 44 days after first head impact (Fig. 3A). As shown in Figures 3C to 3E, whether the RGC count was averaged between both eyes, or determined separately in the right and left eye, the number of RGCs averaged from fields representing all retinal regions in the TBI group was significantly lower than that of control mice. RSV treatment significantly reduced RGC loss in the TBI group compared to the PBS-treated TBI group. The left eyes of RSV-treated TBI mice also had significantly higher RGC numbers than those of PBS-treated TBI mice (see Fig. 3D), whereas RGC counts showed a nonsignificant trend toward an increase in the right eyes of the RSV-treated TBI group. 

Figure 3.

 

Pharmacologic activation of SIRT1 attenuates RGC loss. (A) Representative images of Brn3a-labeled flat-mounted retinas of mice from control (no TBI), TBI, and TBI + RSV groups. (B) Diagram shows 12 standardized fields imaged in each retina. (C–E) Aggregate RGC counts across all retinal regions of both eyes, and right and left eyes separately. Average RGC count in both eyes dropped significantly (*P < 0.05) in the TBI group (n = 6) compared with controls (n = 3). TBI + RSV mice (n = 3) showed a significant (*P < 0.05) improvement in the number of RGCs compared with TBI mice. The right eyes of TBI mice demonstrated significantly (**P < 0.01) reduced numbers of RGCs compared with control mice. RSV treatment induced a nonsignificant trend toward improved RGC numbers. The left eyes of TBI mice demonstrated a significantly (***P < 0.001) reduced number of RGCs compared with control mice. TBI + RSV mice showed a significant (*P < 0.05) improvement in the number of RGCs compared with TBI mice. (F, G) RGC counts in central, mid-peripheral, and peripheral retina. RSV significantly attenuated RGC numbers in TBI + RSV mice (**P < 0.01) mice compared with TBI mice in the central retina of the left eyes. RSV significantly attenuated RGC numbers in TBI + RSV mice compared with TBI mice in the mid-peripheral and central retinas of the right eyes. (H) Representative images of neurofilament-labeled sections of optic nerves of mice from control (no TBI), TBI, and TBI + RSV groups. (I) TBI mice demonstrated significantly (**P < 0.01) lower mean fluorescence signal values of neurofilament staining compared with control mice. TBI + RSV mice showed a significant (**P < 0.01) improvement in mean fluorescence signal values of neurofilament staining compared with TBI mice. RGC counts and neurofilament staining were compared by 1-way ANOVA. Data from one of three representative experiments are shown.

We further determined whether RSV treatment equally or unequally affected all regions of the retina by analyzing the number of surviving RGCs in the peripheral, mid-peripheral, and central retina. Figure 3B shows the 12 standardized fields analyzed. The RGCs counted in the peripheral retina included those in fields 1, 6, 7, and 12. The mid-peripheral retina consisted of fields 2, 5, 8, and 11. The central retina consisted of areas 3, 4, 9, and 10. Each region in TBI mice showed a significant decrease in RGC numbers compared with the same region in control mice in either the left or right eyes, indicating that TBI affected all regions of the retina indiscriminately (see Fig. 3F). The number of RGCs in the central retina of RSV-treated TBI mice was significantly higher than that of PBS-treated TBI mice in both the left and right eyes. Similarly, the number of RGCs in the mid-peripheral retina of the left eyes was higher in RSV- versus PBS-treated mice. Similar to effects on RGC numbers, PBS-treated TBI mice had significantly reduced neurofilament staining of RGC axons in the optic nerve as compared to control mice, and RSV treatment prevented this loss of RGC axons (see Figs. 3H, 3I). 

Effects of TBI on Visual Function in Mice With Constitutive Overexpression of SIRT1

RSV is known to increase SIRT1 activity, suggesting upregulation of SIRT1 might exert similar effects as RSV treatment.²⁹ To assess this, we administered TBI to SIRT1 overexpressing mice. We used hemizygous transgenic SIRT1 KI mice in which SIRT1 cDNA was expressed into the β-actin locus. Mice that are hemizygous for this transgene express normal levels of β-actin and higher levels of SIRT1 protein in all tissues.²⁵ Mice were randomly assigned into 3 groups: (1) a control uninjured group of WT C57BL/6J mice, (2) a TBI group of WT mice, and (3) an SIRT1 KI TBI group. As shown in Figure 4A, the RR times measured after each impact in the TBI and SIRT1 KI TBI groups showed a significantly higher RR time than the control uninjured group. The RR times were not statistically different between the WT TBI and SIRT1 KI TBI groups. Thus, impacts induced similar degrees of TBI with or without overexpression of SIRT1. 

Figure 4.

 

SIRT1 overexpression in TBI mice did not affect RR time but improved visual function. (A) The RR time did not differ significantly between WT TBI and SIRT1 KI TBI mice, and both were higher than control mice. (B–D) When averaged between both eyes, or measured separately in right and left eyes, OKR scores compared by repeated measures ANOVA show a significant (***P < 0.001) progressive decrease over a 6-week time course in TBI mice (n = 4) compared with control (n = 4) mice. SIRT1 overexpression significantly attenuated vision loss in SIRT1 KI TBI mice (n = 4, ***P < 0.001) compared with WT TBI mice. There was no significant difference in visual function between SIRT1 KI TBI and control WT mice. Data from one of three representative experiments are shown.

Similar to WT mice treated with RSV, SIRT1 KI mice also exhibited significantly higher OKR scores for both eyes after TBI (i.e. preserved visual function; Fig. 4B). Repeated measures ANOVA showed a significant decrease in OKR scores across a 6-week period averaged between both eyes of TBI mice (P < 0.001) compared with control mice. In contrast, the OKR scores of SIRT1 KI TBI mice were significantly (P < 0.001) improved compared to the WT TBI mice. There was no significant difference between SIRT1 KI TBI and control groups. The OKR scores of the left and right eyes were again plotted separately (see Figs. 4C, 4D). There was a significant decrease (P < 0.001) in OKR scores for both the right and left eyes in TBI mice compared with controls, and SIRT1 KI TBI mice showed a significant increase in OKR scores compared with the TBI group. 

Effects of TBI on RGC Survival in Mice Constitutively Overexpressing SIRT1

RGC loss was determined in flat mounted preparations of the retinas of control WT, WT TBI, and SIRT KI TBI mouse groups at day 44 after the first impact (Fig. 5A). The TBI group showed a significant (P < 0.001) bilateral decrease in RGC survival compared with the control group (see Figs. 5B–D). In contrast, SIRT1 KI TBI mice showed a significant (P < 0.05) bilateral increase in RGC numbers compared to the WT TBI mice. Further analysis showed that RGC loss due to TBI not only occurred bilaterally, but also in the central, mid-peripheral, and peripheral regions of the retina (see Figs. 5E, 5F). Every region of the retina of TBI mice showed a significant (P < 0.001) decrease in RGC numbers compared with the same corresponding region in control mice. Interestingly, RGC loss in SIRT1 KI TBI mice was significantly (P < 0.001, P < 0.01, and P < 0.05) attenuated in all regions of the retina compared with WT TBI mice. Similar to the effects on RGC numbers, PBS-treated TBI mice had significantly reduced neurofilament staining of RGC axons in the optic nerve as compared to control mice, and this loss of RGC axons was significantly attenuated in SIRT KI TBI mice (see Figs. 5G, 5H). 

Figure 5.

 

SIRT1 overexpression attenuates RGC loss in mice subjected to TBI. (A) Representative images of Brn3a-labeled flat-mounted retinas of mice from control (no TBI), TBI, and SIRT1 KI TBI mouse groups. (B) TBI mice (n = 4) demonstrated a significantly (***P < 0.001) reduced number of RGCs (averaged across all retinal regions and averaged between both eyes) compared with control mice (n = 4). SIRT1 KI TBI (n = 4) mice showed a significant (*P < 0.05) improvement in the number of RGCs compared with TBI mice. (C) The right eyes of TBI mice demonstrated a significantly (***P < 0.001) reduced number of RGCs compared with the right eyes of control mice. SIRT1 overexpression significantly (*P < 0.05) preserved the number of RGCs compared with TBI mice. (D) The left eyes of TBI mice demonstrated a significantly (***P < 0.001) reduced the number of RGCs compared with control mice. SIRT1 overexpression significantly (*P < 0.05) preserved the number of RGCs compared with TBI mice. (E, F) RGC counts in central, mid-peripheral, and peripheral retina of control, TBI and SIRT1 KI TBI mice. SIRT1 overexpression significantly increased RGC numbers in the SIRT1 KI TBI mice compared to TBI mice in the central, mid-peripheral, and peripheral retina of both the right and left eyes (***P < 0.001; **P < 0.01; and *P < 0.05). (G) Representative images of neurofilament-labeled sections of optic nerves of mice from control (no TBI), TBI, and SIRT1 KI TBI mouse groups. (H) TBI mice demonstrated significantly (*P < 0.05) lower mean fluorescence signal values of neurofilament staining compared with control mice. SIRT1 KI TBI mice showed a significant (*P < 0.05) improvement in mean fluorescence values of neurofilament staining compared with TBI mice. RGC counts and neurofilament staining were compared by 1-way ANOVA. Data from one of three representative experiments are shown.

Discussion

In indirect TON induced by TBI, blunt trauma is transmitted through the skull to the region of the optic canal which is particularly susceptible to shock injury.² The controlled intensity of the impacts on the skull used in the current studies precludes more severe injuries, including bone fracture, stroke, hemorrhage, and brain and spinal cord injuries.²⁴ The RR time in TBI mice, which is presumed to be a surrogate for a human regaining consciousness after a traumatic impact to the skull,³⁰ was significantly higher after the first impact but decreased progressively closer to that of control uninjured mice after the fifth impact. Although this observation suggests a form of adaptability to a blunt trauma, it does not necessarily indicate absence of short- and long-term neuronal and functional damages. In the current study, the RR times observed between PBS-treated TBI mice and TBI mice treated with RSV or TBI mice overexpressing SIRT1 were similar, suggesting that a similar degree of brain injury was induced in each group, and is unaffected by SIRT1. Therefore, effects of RSV and SIRT1 overexpression on visual function and RGC survival likely reflect neuroprotective effects on stressed RGCs, as opposed to reduction of the amount of initial injury. 

Direct mechanical trauma to the skull causes immediate tearing and shearing of RGC axons, which together with some degree of optic nerve swelling compromises blood supply to RGCs, setting up a downward cascade toward death of RGCs (and potentially other neuronal cells as well) and subsequent vision deterioration/loss.^31,32 Whereas several models of indirect TON have been used to recapitulate effects on RGCs,² including the blunt head impact TBI model²⁴ used in the current studies, the smaller size of the optic canal and overall smaller skull size in mice than humans may present a limitation in how similarly these forces are distributed in the mice. The degree to which forces are disseminated differently in mice is not well understood, but nonetheless modeling indirect TON in mice has been described as a useful model for understanding the extent of neuronal loss.² 

One of the objectives of this study was to test the pharmacological potential of RSV and its ability to prevent RGC loss in TBI-induced optic neuropathy. RSV was administered through the intranasal route, as previous studies have shown that the efficiency of the nose-to-CNS delivery route of drugs can surpass the intradermal or oral administration.³³ The intranasal delivery route has a significant clinical potential as it is a simple and noninvasive drug administration route with rapid delivery to nervous system tissue and limited systemic exposure.³⁴ A study by Who et al. showed that intranasal administration of RSV improved learning and memory in animals, as well as remission of neuroinflammation via inhibition of interleukins.³⁵ However, our data demonstrated that whereas RSV administration minimally, if at all, affected the RR time, it significantly reduced vision dysfunction in both eyes of the TBI mouse group compared to the PBS-treated TBI group. RSV administration was also associated with reduced loss of RGCs, suggesting that visual improvement stems directly from RSV-mediated RGC survival. This is consistent with the therapeutic benefits of RSV, which has been shown to have a wide range of protective effects in the eyes against ischemic retinal damage, primary open angle glaucoma, macular degeneration, and diabetic retinopathy.^36–39 Molecularly, RSV has diverse biological activities, including antioxidant, anti-inflammatory, and antiapoptotic actions that provided neuroprotective effects in animals subjected to different types of acute injuries to the brain and spinal cord,⁴⁰ potentially by its demonstrated ability to antagonize the overproduction of ROS and activation of inflammatory factors.⁴¹ However, despite its promising potential in harnessing oxidative and inflammatory reactions, RSV is a poor direct antioxidant and anti-inflammatory agent, less potent than antioxidants like vitamin C and cysteine.⁴² 

There is experimental evidence to suggest that although RSV has multiple targets,^43,44 its neuroprotective effects are mediated by SIRT1.^45,46 Our data showed that overexpression of SIRT1 in mice recapitulated the neuroprotective action of RSV. SIRT1 is known to be activated by RSV,⁴⁷ and many of its effects are consistent with deacetylation/activation of SIRT1 targets, such as PGC-1α,⁴⁸ which increases ROS-detoxifying enzymes and enhances mitochondrial biogenesis and function.⁴⁹ Consistent with this, SIRT1 activators significantly reduced ROS levels and increased mitochondrial metabolism in different models of optic neuropathies.^50,51 

Conclusions

Our data demonstrate that intranasal administration of RSV can reduce RGC death and preserve visual function in the mouse model of traumatic optic neuropathy associated with TBI, and that overexpression of SIRT1 was able to mimic the RSV effects. These findings expand the therapeutic potential of RSV to a key model that mimics features of blunt head force induced TON observed in human patients and results further support RSV's recognized benefit as a pro-survival anti-cell death factor. The potential beneficial effects of RSV in managing optic neuropathy caused by TBI in humans warrant further investigation. 

Acknowledgments

The authors thank Jipeng Yue for his helpful contribution to cell counting, and they thank Miranda Meng and Jacob Rossman for their help with neurofilament staining and quantification. 

Supported by the National Institutes of Health Grants (EY019014 and EY301163), the Department of Defense (USAMRAA) award number HT9425-23-1-0725, the RWJ-Harold Amos Faculty Development Award, the Linda Pechenik Montague Investigator Award, the Dean's Innovation Fund, the Research to Prevent Blindness, the Paul and Evanina Mackall Foundation Trust, the Center for Advanced Retinal and Ocular Therapeutics, and the F. M. Kirby Foundation. 

Disclosure: A. Kwok, None; B. Chaqour, None; R.S. Khan, None; P. Aravand, None; K. Dine, None; A.G. Ross, None; K.S. Shindler, None