Spinal stiffness assessment by shear wave imaging in a genetic zebrafish scoliosis model treated with anti-inflammatory medication
Authors: Gopaul Josh , Van Gennip Jenica , Baranger Jerome , Griffiths Nigel , Ciruna Brian , Villemain Olivier 
Published: May 2023

Background: Idiopathic scoliosis (IS) is a disease characterized by an abnormal lateral curvature of the spine that affects up to 4% of the population. Recent models in zebrafish have elucidated genetic variants which contribute to scoliotic curvature through mechanisms of neuroinflammation, and oxidative stress. Additionally, in-vivo clinical studies have demonstrated that spinal structures stiffen in patients with IS. Shear wave elastography (SWE) is novel, non-invasive imaging technique used to quantitatively estimate tissue stiffness. Objective: Here, we investigated spinal stiffness using SWE in genetically mutant zebrafish with a heterozygous scoliosis variant ( dmh4/+) treated with N-acetylcysteine ethyl ester (NACET), a potent anti-oxidant and anti-inflammatory medication which has been demonstrated to reduce scoliosis curvature formation in zebrafish by up to 70%. Hypothesis: Compared to wildtype, spinal stiffness will increase in dmh4/+ zebrafish and there will be no difference in stiffness for NACET treated zebrafish. Methods: Wildtype (WT; n=20) and mutant ( dmh4/+; n=20) zebrafish aged 10 days post fertilization (dpf) (n=10 & n=10 treated with 500 μM of NACET), 16 dpf (n=10), and 21 dpf (n=10) were sacrificed and submerged in a dish containing 2% low melt agarose gel. An 18.75 MHz linear ultrasound probe was clamped to the dish apparatus dorsal to the zebra fish. Spinal stiffness was assessed by SWE, where faster shear wave velocities (SWV) represent stiffer tissue. The SWV data was processed using a well-verified, custom SWE analysis program on MATLAB by a n operator blinded to the zebrafish genotypes. Results: The spinal curvature of dmh4/+ zebrafish aged 10 dpf were morphologically indistinguishable from WT zebrafish aged 10 dpf. SWV was significantly increased in dmh4/+ zebrafish aged 10 dpf compared to WT (p<0.05). There was no difference in SWV between NACET treated dmh4/+ and NACET treated WT zebrafish aged 10 dpf (p>0.05). Furthermore, NACET treated dmh4/+ and WT zebrafish aged 10 dpf exhibited no difference in SWV compared to untreated WT zebrafish aged 10 dpf (p>0.05). Additionally, SWV was significantly increased in dmh4/+ zebrafish aged 16 and 21 dpf compared to WT (p<0.05). Conclusion: Here we demonstrate that spinal stiffness increases in zebrafish with scoliosis before the curvature pheno type is present and this increase in stiffness is sustained throughout development in later ages. Additionally, spinal stiffness in scoliosis is reduced following treatment with NACET. Our results provide evidence to support clinical trials assessing stiffness as preclinical diagnostic criteria for IS in children. Biotalent Canada - public grant This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.