Supplementary Material for: Detecting DWI-Negative Strokes in Rat Models of Acute Internal Carotid Artery Occlusion using Magnetic Resonance Spectroscopy

Objective: To explore the potential of Magnetic Resonance Spectroscopy (MRS) for the early diagnosis of the ischemic brain tissue with negative Diffusion Weighted Imaging (DWI) results in the model of acute internal carotid artery occlusion (AICAO) in rats. Methods: Fifteen rats were randomly assigned to three groups: the sham group (SHAM, n=5), the 2-hour ischemia model group (CI2H, n=5), and the 12-hour ischemia model group (CI12H, n=5). In the experimental group, the method of acute internal carotid artery occlusion was adopted to establish a right cerebral ischemia model. After the successful establishment of the model, DWI and MRS scans were first performed, and then the MRS results were recorded. Immunohistochemical analysis of zonula occludens-1 (ZO-1), neuron-specific enolase (NSE), S-100β protein, and cytochrome C (CytC) was conducted on paraffin-embedded brain sections, followed by Spearman correlation analysis between relevant parameters and these proteins. Results: Lipid (Lip) levels were lower in the SHAM group compared to the CI2H group (P<0.05), but no significant difference was observed between the SHAM and CI12H groups. Lip was negatively correlated with ZO-1 across all groups (ρ=-0.768, P<0.001). Lactate (Lac) levels were lower in the SHAM group than in the CI2H and CI12H groups (P<0.05), and Lac was positively correlated with CytC across all groups (ρ=0.801, P<0.001). Apparent Diffusion Coefficient (ADC) values on the right side of the brain were lower in the CI12H group than in the SHAM group (P<0.05), with no significant difference compared to the CI2H group.The level of N-acetyl aspartate (NAA) in the CI12H group was lower than that in the SHAM group and the CI2H group (P < 0.05), and there was no statistically significant difference in the level of NAA between the CI2H group and the SHAM group. The Cho level in the CI12H group exhibited a statistically significant increase compared to both the SHAM and CI2H groups (P < 0.05), and there was no statistically significant difference in the level of Choline (Cho) between the CI2H group and the SHAM group. Conclusion: Brain damage in AICAO rats became evident at two hours after successful modeling. MRS detected brain damage earlier than DWI, with Lac and Lip as the most sensitive markers, preceding changes in NAA, Cho, and ADC values.