(H-301) Sex and Age Comparison of Neck Vasculature and Brain Biochemistry in Mice Model

Introduction:: For the brain to properly function it must receive an adequate blood supply from the carotid artery and blood removal through the jugular vein. Changes in the carotid arteries and jugular veins can influence flow pulsatility and pulsatile pressure in the microvasculature of the brain, potentially leading to cognitive decline¹. Sex and age both impact changes in vasculature, such as increased stiffness. An increase in vascular stiffening is predominantly due to aging, however, post-menopausal women have a higher incidence than age-matched men². Additionally, sex and age can impact brain biochemistry³. Studying how changes in vasculature affect the brain, across sex and age groups, may give insight to the formation of common neurological disorders. To analyze brain changes, it is important to look at the brain biochemistry. Mass spectrometry is a technique capable of ionizing molecules on the surface of a tissue sample to collect information on the spatial intensity of biomolecules and has been widely used for brain omics studies⁴. Lipids are the basic component of cell membranes and therefore, play a vital role in brain health and function⁵. The goal of this research is to understand how and if changes in vasculature alter the brain biochemistry and if there are variations across sex and age. Understanding the relationship between vasculature and brain biochemistry in a healthy state, will allow for further analysis of their relationship in certain disease states.


Materials and Methods:: Young and aged male and female C57BL6 mice were imaged via ultrasound (Vevo 3100, Visual Sonics). Images were collected in B-mode and power Doppler mode to measure the diameter of the carotid arteries and jugular veins as well as the velocity of blood moving through them in systole and diastole. These values were used to calculate the circumferential cyclic strain and the wall shear stress of the carotid and jugular. Using JMP, the statistical analysis was performed using a one-way ANOVA test. Once imaged, one group of mice were sacrificed via conscious decapitation for quick removal of the brain and then flash frozen on liquid nitrogen. The other group of mice were sacrificed via isoflurane overdose in order to preserve the carotid arteries and jugular veins. Once the carotid, jugular, and brain were removed, they were flash frozen on liquid nitrogen. Once frozen, the tissue was stored in a -80 °C freezer. The carotid, jugular, and brain tissues were sectioned onto slides with a cryostat. After sectioning, the slides were placed in a desiccator in preparation for MALDI MSI. Two matrices, CHCA and 9AA, were sprayed on the slides using an HTX sprayer before running in positive/negative ion mode in mass spectrometry. Once the slides were imaged, they were stained, and a coverslip was mounted in preparation for imaging using a microscope. Mass spectrometry data was analyzed to determine the lipids present in the tissue.







Results, Conclusions, and Discussions:: From the ultrasound B-mode images, the diameter of the jugular veins and carotid arteries in systole and diastole were measured using Vevo software. Using the pulse doppler data, the average velocity of blood moving through the carotid and jugular were measured. The circumferential cyclic strain (CCS) and wall shear stress (WSS) were calculated for the carotid and jugular. There were no statistically significant differences between male and female or young and old mice for the CCS or WSS. However, the jugular average velocity and diameter between age-matched male and female were statistically significant with p-values of 0.03 and 0.009. Figure 1 shows the average circumferential cyclic strain for N=5 young female, N=5 young male, and N=5 old male. Figure 2 shows the jugular average diameter for N=5 male and N=5 female age-matched mice. The brain tissue will be analyzed to see if these statistically significant differences in the vasculature correlate to differences in the brain biochemistry. The brain biochemistry is expected to be different between the group of mice that were sacrificed via isoflurane overdose versus those sacrificed via decapitation.
The goal of this research is to determine whether changes in vasculature correlate to differences in brain biochemistry. Using mass spectrometry and ultrasound data, the carotid arteries and jugular veins will be analyzed to look for possible differences. The brain tissue will be analyzed to look for differences that may be related to possible differences seen in the vasculature. From the ultrasound data collected, there are statistically significant differences between male and female for the jugular average velocity and diameter. In the female group, the average jugular velocity and diameter were larger than the male group. Analyzing the biochemistry of this brain tissue may show differences related to those seen in the vasculature. Understanding the relationship between vasculature and brain biochemistry in healthy conditions, will lay the groundwork for understanding the effect of stiff vasculature.
Future work will consist of analyzing the vasculature and brain biochemistry of young and aged mice groups with stiff vasculature to analyze the relationship between stiff vasculature and the brain biochemistry across sex and age.




Acknowledgements (Optional): : Dr. Colleen Crouch and the Crouch Imaging Lab


References (Optional): : 1. Cooper, L. L. et al. Pulse 4, 69–77 (2016) 2. DuPont, J. J. et al. Br J Pharmacol 176, 4208–4225 (2019) 3. Winder, N. R. et al. Am J Physiol Heart Circ Physiol 320, H424–H431 (2021) 4. Xu, G., Li, J., Jianjun Li, C., & Health Therapeutics, H. (2018) 5. Kao, Y. C., Ho, P. C., Tu, Y. K., Jou, I. M., & Tsai, K. J. (2020)