Stroke promotes the development of brain atrophy and delayed cell death in hypertensive rats

Abstract

Post-stroke cognitive impairment (PSCI) is a major source of disability, affecting up to two thirds of stroke survivors with no available therapeutic options. The condition remains understudied in preclinical models due to its delayed presentation. Although hypertension is a leading risk factor for dementia, how ischemic stroke contributes to this neurodegenerative condition is unknown. In this study, we used a model of hypertension to study the development of PSCI and its mechanisms. Spontaneously hypertensive rats (SHR) were compared to normotensive rats and were subjected to 1-h middle cerebral artery occlusion or sham surgery. Novel object recognition, passive avoidance test and Morris water maze were used to assess cognition. In addition, brain magnetic resonance images were obtained 12-weeks post-stroke and tissue was collected for immunohistochemistry and protein quantification. Stroked animals developed impairment in long-term memory at 4-weeks post-stroke despite recovery from motor deficits, with hypertensive animals showing some symptoms of anhedonia. Stroked SHRs displayed grey matter atrophy and had a two-fold increase in apoptosis in the ischemic borderzone and increased markers of inflammatory cell death and DNA damage at 12 weeks post-stroke. This indicates that preexisting hypertension exacerbates the development of secondary neurodegeneration after stroke beyond its acute effects on neurovascular injury.

Figure 1

Hypertension increases stroke mortality and reduces spontaneous recovery. (A) Experimental design showing different time points for behavioral testing. (B) The mean arterial blood pressure of a cohort of SHR animals of the same age and weight of our stroke animals was followed for 2 weeks to establish their blood pressure (n = 6). (C) 63% of the Wistar animals achieved complete spontaneous post-stroke recovery after 24 h of MCAO, compared to 0% for SHR animals. (P < 0.0001, Fischer’s exact test) (D) & (E) Body weight was recorded for 12 weeks following MCAO comparing sham animals from both strains to stroke animals (n = 6–12 per group).

Figure 2

Hypertension causes slower sensorimotor recovery. (A) Both strains had significant neurological impairment (Modified Bederson’s Score) starting 24 h after stroke and up to day 14. (n = 6–12 per group) (One sample t-test compared to the maximum score of 8). (B) There was no significant difference between the 4 groups in object discrimination in the novel object recognition test at 2- and 12-weeks post MCAO. (n = 6–12 per group). (C) SHRs showed a trend for reduced total object exploration time at 2-weeks post stroke (p = 0.058, 2-way ANOVA). The difference disappeared at 12-weeks post-stroke. (n = 6–12 per group).

Figure 3

Stroke causes long-term fear associated memory dysfunction and hypertension induces behavioral despair. (A) Time to reach the platform during the acquisition of the Morris Water Maze was recorded. All groups showed a consistent learning curve (P for time < 0.001, 2-way ANOVA). (B) Average swim speed over the course of the experiment was measured for all groups. (P > 0.05, 2-way ANOVA) (n = 6–12 per group). (C) Time spent in the platform zone and (D) time spent in the target quadrant were measured in the probe trial to examine the memory function (P > 0.05, 2-Way ANOVA). (E) Total distance travelled during the probe test was a measurement for behavioral despair and depression. (P for strain < 0.001, 2-way ANOVA). (a,b: Tukey post-hoc multiple comparisons, pairs of means with different letters are significantly different) (F) Passive Avoidance showed a decrease in the average latency to enter the shock arm for stroked animals. (P < 0.01, 2-way ANOVA) (n = 6–12 per group).

Figure 4

Stroke induces enlarged ventricles and grey matter atrophy in SHRs, as measured by MRI at 12-weeks post-stroke. (A) Representative images for diffusion weighted MRI, week 12 post-stroke. White indicates areas with high water content. (B) Ischemic tissue as % of the volume of the contralesional hemisphere (P > 0.05, t-test) (n = 6–12 per group). (b) Total volume of lateral ventricles as % of total brain volume, with SHR showing significant bilateral enlargement of lateral ventricles (P for strain < 0.01, 2-way ANOVA). (D) Volume of the ipsilesional ventricle as % of the contralesional ventricle, with significant enlargement for stroked animals in both strains. (P for surgery < 0.001, 2-way ANOVA). (E) Total volume of the ipsilesional hemisphere as % of total brain volume, decreased for stroked animals in both strains. (P for surgery < 0.001, 2-way ANOVA). (a,b,c: Tukey post-hoc multiple comparisons, pairs of means with different letters are significantly different).

Figure 5

Stroke induces neuronal apoptosis in the ischemic border zone region of brain tissue, which is exacerbated by hypertension. (A) Representative image of the positions from which the TUNEL and IBA1 images were taken. The polygon outline represents the ischemic tissue, the squares represent fields for quantitation. (DAPI) (B) SHRs displayed a significant increase in apoptosis (P < 0.05, 2-way ANOVA) compared to normotensive animals. Stroked animals displayed a significant increase in apoptosis (P < 0.01, 2-way ANOVA) compared to Sham animals. (n = 4–8 per group). (C) Stroked animals displayed a significant increase in IBA1 staining compared to shams (P < 0.001, 2-way ANOVA) (n = 4–8 per group). (a,b: Tukey post-hoc multiple comparisons, pairs of means with different letters are significantly different.) (D) Representative images from TUNEL staining. (E) Representative images from IBA1 staining (false color).

Figure 6

SHRs display increases in markers of neuronal damage following stroke that are not increased in normotensive animals. (A) Representative image for Western blot data. (B) Stroked SHR show increase in HMGB1 (p (interaction) < 0.05, 2-way ANOVA). (n = 4–8, per group). (C) and (D) Stroked SHRs show increase in activated MMP9 (p (interaction) < 0.01 for upper band and < 0.001 for lower band, 2-way ANOVA). Stroked SHRs show an increase of activated MMP9 (p < 0.05 for upper band and < 0.01 for lower band, Bonferroni post-hoc test) (n = 4–8, per group). (E) SHR show increase in full-length PARP1 after stroke (p (interaction) < 0.01, 2-way ANOVA). Stroked SHR show an increase of PARP1 compared to stroked Wistars (p < 0.05, Bonferroni post-hoc test) (n = 4–8, per group). (a,b: Tukey post-hoc multiple comparisons, pairs of means with different letters are significantly different).

Figure 7

Stroke induces long term neurodegeneration in hypertensive animals. Ischemic stroke causes a marked inflammation in stroked animals. In hypertensive animals, which already suffer from an increase in ROS production, the ischemic insult and the oxidative stress result in chronic increase in DNA damage and neuronal cell death, marked with an increase in PARP1 and HMGB1. PARP1 increase induces the transcription of MMP9, which triggers apoptosis. The result of these processes is chronic neuronal loss in the form of grey matter atrophy.