Short-term preoperative dietary restriction is neuroprotective in a rat focal stroke model

Abstract

Stroke is a major complication of cardiovascular surgery, resulting in over 100,000 deaths and over a million postoperative encephalopathies annually in the US and Europe. While mitigating damage from stroke after it occurs has proven elusive, opportunities to reduce the incidence and/or severity of stroke prior to surgery in at-risk individuals remain largely unexplored. We tested the potential of short-term preoperative dietary restriction to provide neuroprotection in rat models of focal stroke. Rats were preconditioned with either three days of water-only fasting or six days of a protein free diet prior to induction of transient middle cerebral artery occlusion using two different methods, resulting in either a severe focal stroke to forebrain and midbrain, or a mild focal stroke localized to cortex only. Infarct volume, functional recovery and molecular markers of damage and protection were assessed up to two weeks after reperfusion. Preoperative fasting for 3 days reduced infarct volume after severe focal stroke. Neuroprotection was associated with modulation of innate immunity, including elevation of circulating neutrophil chemoattractant C-X-C motif ligand 1 prior to ischemia and suppression of striatal pro-inflammatory markers including tumor necrosis factor α, its receptor and downstream effector intercellular adhesion molecule-1 after reperfusion. Similarly, preoperative dietary protein restriction for 6 days reduced ischemic injury and improved functional recovery in a milder cortical infarction model. Our results suggest that short-term dietary restriction regimens may provide simple and translatable approaches to reduce perioperative stroke severity in high-risk elective vascular surgery.

Figure 1. Preoperative 3-day water-only fasting is neuroprotective against stroke.

(A) Experimental timeline indicating periods of ad libitum feeding and fasting relative to the onset of tMCAO on day 0. AL, ad libitum fed (n = 11); FA, fasted (n = 14). (B) Average body weights prior to and after tMCAO; F_{8, 23} = 17.69, ***p<0.0001, 2-way ANOVA. (C) Blood glucose levels on the indicated days prior to and after tMCAO; F_{2, 23} = 14.09, ***p<0.0001, 2-way ANOVA. (D) Body temperature before tMCAO and 1 hour after reperfusion; ***/^###p<0.001, Student's t-test. (E) Infarction volumes at d7 after tMCAO; *p = 0.0215, Student's t-test. (F) Representative MRI images of the lesioned brain sections with green lines surrounding the lesion.

Figure 2. Molecular mechanisms of fasting-induced neuroprotection.

(A) Experimental timeline indicating dietary treatments and experimental endpoints relative to tMCAO on day 0. (B) Relative expression of the indicated genes in the striatum of ad libitum fed (AL, n = 6) and fasted (FA, n = 5) rats at baseline, measured by qPCR and expressed relative to the AL group. (C) Relative expression of selected genes in AL (n = 5) and FA (n = 6) rats 24 hours after tMCAO in the unlesioned left (L) and lesioned right (R) striata, measured by qPCR and expressed relative to the unlesioned AL group; *p<0.05, **p<0.01, ***p<0.001, 1-way ANOVA. (D) Serum CXCL1 levels in AL (n = 12) and FA (n = 12) rats at baseline; **p = 0.0021, Student's t-test.

Figure 3. Protein-free DR is neuroprotective against stroke.

(A) Experimental timeline indicating periods of ad libitum access to a complete diet (AL, n = 15) or restricted access to a protein-free diet (PF, n = 14) relative to the onset of tMCAO on day 0. (B) TTC-stained brain sections showing infarct size (white area). (C) Total infarction volume on d2 after tMCAO; *p = 0.0396, Student's t-test. (D) Average maximal infarction area from the slice with the largest infarction area per animal; *p = 0.0320, Student's t-test. (E) Biased body swing activity in 20 trials; **p = 0.0016, Mann-Whitney U-test. (F) Behavioral performance assessed by modified Bederson's score; *p = 0.0396, Mann-Whitney U-test.

Figure 4. Protein-free DR promotes functional recovery after stroke.

(A) Experimental timeline indicating periods of ad libitum access to a complete diet (AL, n = 14) or restricted access to a protein-free diet (PF, n = 14) relative to the onset of tMCAO on day 0 and subsequent behavioral testing on days 2, 7 and 14. (B) Average body weights on the indicated days relative to tMCAO on day 0; F_1,26 = 96.20, ***p<0.0001, 2-way ANOVA. (C-G) Behavioral tests on the indicated days after tMCAO: (C) horizontal activity; F_1,26 = 4.994, *p = 0.034, 2-way ANOVA; (D) vertical activity; F_1,26 = 4.150, p = 0.052, 2-way ANOVA; (E) biased body swing activity in 20 trials; *p = 0.0211, Mann-Whitney U-test; (F) modified Bederson's score; **p = 0.0018, Mann-Whitney U-test; (G) cylinder test measured on d14 after tMCAO; *p = 0.0492, Mann-Whitney U-test.