The role of SARM1 in traumatic axonopathy in the mouse visual system.

Youngrim Lee,1 Athanasios S. Alexandris,1 Mohamed Lehar4, Jiwon Ryu,1 Zahra Alam1, Payton Flores1, Vassilis E. Koliatsos1,2,3 Departments of Pathology,1 ,2 and Psychiatry and Behavioral Sciences,3 Otolaryngology-HNS4 | Johns Hopkins University School of Medicine | Baltimore, MD

Introduction & Aims Methods Results (TBI) has been a major cause of adult mortality and permanent Stereological analysis. disability, including cognitive, psychosocial, and physical deficits. One of the main Unbiased and efficient estimation of intact and damaged myelinated in the ON 2. Significant loss and ongoing pathology are present in both genotype groups after TBI. neuropathologies contributing to the permanent disability in TBI patients is traumatic was performed by an investigator blinded to experimental groups, using Stereo The number of intact axons significantly decreases while the number of damaged axons dramatically increases at 21 days axonal injury (TAI). TAI is a primary axonal insult resulted from rotational acceleration of Investigator® software (Microbrightfield Inc., Williston, VT) under 100x magnification. after IA-TBI in both WT and SARM1 KO mice. It is also important to note that the SARM1 KO mice have greater number of the head typically during motor vehicle crashes and high-impact falls. TAI initiates and Systematic random sampling was chosen with the following sampling parameters: intact axons than WT mice at baseline (p = 0.0018). Additionally, a significant loss of total axons is present after IA-TBI in both genotype groups. Bar graphs show means with standard deviation (SD). An independent t-Test was used to determine triggers an axonal program of self-destruction called Wallerian degeneration (WD), counting frame size (4 x 4μm), grid size (17.89 x 17.89μm), sampling fraction (4%). The statistically significant differences between the means in addition to one-tailed p values. A p value <0.05 was considered as total number of axons was automatically estimated by the software (Koschade et al., which results in distal axon fragmentation and degeneration. However, the initiation of statistically significant. WT n = 8 sham, n = 10 IA-TBI; SARM1 KO n = 8 sham, n = 9 IA-TBI. WD requires the activation of Sterile alpha and TIR motif containing 1 (SARM1), which is 2019). a key regulator in a conserved axon death pathway involved in WD (Koliatsos & The criteria for intact and damaged axons: Alexandris, 2019). • Intact axon is featured with organized cytoskeletal structure and compact Thus, this SARM1-mediated axon death pathway has been investigated as a target for . Thickness of myelin is proportional to axon diameter. acute interventions that could mitigate axonal degeneration and preserve neurological • Damaged axon is characterized by collapsed cytoskeleton (darkening of function following TBI. Previous studies have demonstrated the protective effect of axoplasm) and/or abnormal myelin (i.e. myelin thickening, myelin outfolding, SARM1 genetic deletion against axon degeneration in corpus callosum (CC) and disintegration of myelin sheath). corticospinal tract (CST) of mice subjected to injury in a closed head TBI model at early phase post-injury time points (Henninger et al., 2016; Ziogas et al., 2018; Armstrong et al., 2019). In contrast, there was no axonal protection observed in CC of the TBI-induced Results mice at late phase post-injury time point (Armstrong et al., 2019). 1. A significant population of the damaged axons is present in the ON of both Although there has been studies focusing on WD in the optic nerve (ON) stretch-injury WT and SARM1 KO mice on day 21 post IA-TBI injury, indicating the model of TBI (Maxwell et al., 2015), there has not been studies investigating the effect of presence of ongoing degeneration. A and B show the population of the intact axons. 3. SARM1 deletion reduces ongoing pathology afterTBI. SARM1 deletion on axon degeneration after TBI in ON. Therefore, we conducted a On the other hand, C and D show many profiles of the damaged axons, including myelin To determine the protective effect of SARM1 deletion on axon degeneration unbiasedly, it was essential to normalize the baselines of WT and SARM1 KO. Thus, the intact axon survival rate was calculated by comparing to shams per genotype. The stereological analysis to assess the protective effect of SARM1 deletion on traumatic outfoldings (Blue arrows), disintegration of myelin sheath (green arrows), and darkening of intact axon survival rate is higher in SARM1 KO mice than the one in WT mice; however, the difference in the intact axon axonopathy of the mouse ON after impact acceleration TBI in the long term. axoplasm (yellow arrows). All pictures were taken under 100x magnification using a light survival rate between two groups is not statistically significant (p = 0.10). On the other hand, the percentage of damage axons microscope. *The scale bar indicates 50μm. is significantly lower in SARM1 KO mice than the one in WT mice (p=0.022), meaning that SARM1 deletion protects against Materials and Methods WT SARM1 KO chronic pathology after TBI. A B Experimental animals. The subjects of the experiment were 10 weeks old male mice (n=35) and divided into 2 genotype groups: WT (Sarm1+/+) (n=16) and SARM1 KO (Sarm1-/- ) mice (n=19). Subsequently, each genotype group was separated into two SHAM groups: Sham group and injury group exposed to the impact acceleration (IA) method of TBI (IA-TBI group). C D Impact acceleration (IA) model of TBI. IA injury was produced under gas anesthesia with 2% isoflurane. A 5-mm-diameter steel disc was glued on the IA-TBI exposed cranium between bregma and lambda. 50g brass weight was dropped from a height of 85cm through the Plexiglas tube onto the disk glued on mouse that was Conclusions placed prone on a foam bed and secured with tape under References a Plexiglas tube (Xu et al., 2016). 1. Henninger, Nils, et al. “Attenuated traumatic axonal injury and improved functional outcome after traumatic brain injury in mice lacking Sarm1.” • TAI leads to ongoing axon degeneration over the course of weeks on the ON of Brain, vol. 139, 2016, p. 1094-1105. mice after IA-TBI. Tissue preparation and Toluidine blue staining. 2. Koliatsos & Alexandris. “Wallerian degeneration as a therapeutic target in traumatic brain injury.” Curr Opin Neurol. 2019 Dec;32(6):786-795. 3. Koschade, Sebastian E., et al. “Efficient determination of axon number in the optic nerve: A stereological approach.” Experimental Eye Research, Injury group of mice were perfused 21 days post IA-TBI as well as sham mice. ONs were vol. 186, 2019, p. 107710. • The protective effect of SARM1 deletion is absent on primary traumatic dissected from the mice and subsequently, post-fixed with 4% paraformaldehyde and 4. Marion, Christina M., et al. “Sarm1 Deletion Reduces Axon Damage, Demyelination, and White Matter Atrophy after Experimental Traumatic Brain Injury.” Experimental Neurology, vol. 321, 2019, p. 113040. axonopathy on the ON of IA-TBI mice. 0.2% glutaraldehyde, treated with 1% osmium tetroxide, and stained en bloc with 1% 5. Maxwell, William L., et al. “Wallerian Degeneration in the Optic Nerve. Stretch-Injury Model of Traumatic Brain Injury: A Stereological Analysis.” Jouranl of Neurotrauma, Vol. 32, No.11,2015. • Sarm-1 deletion reduces the burden of chronic pathology on the ON at 3 weeks uranyl acetate by order. Then semithin sections (1um) were cut transversely from 6. Xu, Leyan, et al., “Repetitive mild traumatic brain injury with impact acceleration in themouse: Multifocal axonopathy, neuroinflammation, segments of ONs proximal to the optic chiasm. Subsequently, sections were stained andneurodegeneration in the visual system.” Experimental Neurology, Vol. 275, 2016, p.436-449. after TBI. 7. Ziogas, Nikolaos K. & Koliatsos, Vassilis E. “Primary Traumatic Axonopathy in Mice Subjected to Impact Acceleration: A Reappraisal of Pathology with 1% toluidine blue. and Mechanisms with High-Resolution Anatomical Methods.” The journal of Neuroscience , 2018, p. 4031– 4047.