Hyperglycemia Is Associated with Immediate Brain Swelling and Hemorrhagic Transformation After Middle Cerebral Artery Occlusion in Rats

Swelling of the ipsilateral hemisphere was computed using the area of the contralateral and ipsilateral, I _i, slices with

$\mathrm{Swelling}\kern0.24em \mathrm{volume}\;\left(\%\right)=\left(\frac{{\displaystyle \sum}_i\left({I}_i-{C}_i\right)}{\left({\displaystyle \sum}_i{C}_i\right)}\right)100.$

After taking an image of the TTC-stained tissue, the slices were cut into left and right hemispheres. The right hemisphere pieces were combined, flash frozen, and stored at −80 °C. Hemorrhagic transformation was quantified using the hemoglobin assay, as previously described [3, 6]. Hemispheric brain tissue, added to 3 ml PBS, was homogenized for 30 s, followed by sonication on ice for 1 min, and centrifugation at 13,000 rpm and 4 °C for 30 min. After this, 0.2 ml of supernatant was added to 0.8 ml of Drabkin’s reagent (Bio-Rad, Hercules, CA, USA), and allowed to stand in the dark for 15 min at room temperature. Optical density at 540 nm was measured with a spectrophotometer (Genesys 10S, Thermoscientific, Waltham, MA, USA). A standard curve was used to obtain the hemoglobin volume of each sample; error was propagated through all calculations. The standard curve was created by adding known amounts of blood to naïve hemispheric brain tissue, measuring absorbance following the above protocol, and then performing linear regression analysis of the absorbance versus hemoglobin volume.

All measures are presented as the mean ± SEM. Data were analyzed using one-way analysis of variance (ANOVA) with Tukey post hoc tests for the infarct volume, swelling volume, and hemoglobin volume. Blood glucose data were analyzed using one-way repeated measures ANOVA with Tukey post hoc test. A p < 0.05 was considered statistically significant.

Results

Blood glucose of sham and MCAO animals at the beginning of MCAO surgery (1 min pre-MCAO, sham: 242.3 ± 46.4 mg/dl, MCAO: 194.0 ± 22.5 mg/dl) and immediately after reperfusion (5 min post-MCAO, sham: 285.0 ± 46.9 mg/dl, MCAO: 209.3 ± 17.9 mg/dl) were significantly higher than that of sham and MCAO animals 15 min pre-MCAO (15 min pre-MCAO, sham: 75.8 ± 4.3 mg/dl, MCAO: 81.8 ± 2.2 mg/dl) and 24 h post-MCAO (24 h post-MCAO, sham: 75.5 ± 3.3 mg/dl, MCAO: 67.5 ± 3.3 mg/dl) (sham: p < 0.05 vs 15 min pre-MCAO and p < 0.05 vs 24 h post-MCAO for the both the 1 min pre-MCAO and 5 min post-MCAO groups; MCAO: p < 0.05 vs 15 min pre-MCAO and p < 0.05 vs 24 h post-MCAO for the both the 1 min pre-MCAO and 5 min post-MCAO groups) (Table 1).

Table 1

Blood glucose measurements

	Sham	MCAO
15 min pre-MCAO	75.8 ± 4.3	81.8 ± 2.2
1 min pre-MCAO	242.3 ± 46.4*	194.0 ± 22.5*
5 min post-MCAO	285.0 ± 46.9*	209.3 ± 17.9*
24 h post-MCAO	75.5 ± 3.3	67.5 ± 3.3

Blood glucose (mg/dL) was measured before dextrose injection (15 min pre-MCAO), at the beginning of surgery (1 min pre-MCAO), at the beginning of reperfusion (5 min post-MCAO, and 24 h after MCAO (24 h post-MCAO) (n = 5/group))

*p < 0.05 vs 15 min pre-MCAO baseline and 24 h post-MCAO

Progression of Infarct Volume

Within 15 min post-MCAO, the infarct volume (15 min post-reperfusion, 14.9 ± 2.59 %) was significantly higher than sham animals (0.3 ± 0.30 %; p < 0.05), and reached its maximum. No difference in infarct volume was observed between any of the groups after 15 min (p > 0.05 for all group comparisons between 15 min, 1, 3, and 24 h post-reperfusion) and all were significantly higher than sham animals (p < 0.05 vs sham for all groups). The infarct volumes were 10.75 ± 0.79 %, 11.8 ± 3.53 %, and 13.1 ± 1.81 % for the 1, 3, and 24 h post-reperfusion, respectively (Fig. 1).

Fig. 1

Development of infarct volume after MCAO. Infarct volume (%) time course after MCAO. * p < 0.05 vs sham. n = 5/group

Progression of Ipsilateral Hemisphere Swelling

Swelling of the ipsilateral hemisphere was elevated at 15 min (4.3 ± 1.34 %) and 1 h post-reperfusion (6.2 ± 1.50 %) but not significantly different from sham (−0.7 ± 0.66 %) (p > 0.05 vs sham for the 15 min and 1 h post-reperfusion groups). By 3 h after reperfusion, ipsilateral hemisphere swelling reached statistically significant values (9.7 ± 1.91 %; p > 0.05) and continued to increase to the 24 h time point (14.5 ± 2.69 %; p < 0.05 vs sham for 3 and 24 h post-reperfusion groups). At 24 h, the ipsilateral swelling was also significantly higher than the 15 min and 1 h post-reperfusion time points (p < 0.05 vs 15 min and 1 h post-reperfusion) (Fig. 2a).

Fig. 2

Development of brain swelling and hemorrhagic transformation after MCAO. (a) Ipsilateral hemispheric swelling (%) time course after MCAO. *p < 0.05 vs sham. #p < 0.05 vs 15 min. †p < 0.05 vs 1 h. n = 5/group. (b) Hemoglobin volume (μL) describes hemorrhagic transformation after MCAO. *p < 0.05 vs sham. #p < 0.05 vs 15 min. n = 5/group, † p < 0.05 vs 1 h. n = 5/group

Development of Hemorrhagic Transformation

The amount of hemoglobin in the brain tissue was not significantly elevated 15 min after reperfusion (15 min post-reperfusion, 2.0 ± 1.33 μl) compared with sham (0.4 ± 1.03 μl) (p > 0.05). However, 1 h post-reperfusion, the hemoglobin volume (1 h post-reperfusion, 7.7 ± 1.31 μl) was significantly higher than sham and continued to rise to the 3 (9.0 ± 1.90 μl) and 24 h (12.3 ± 2.15 μl) time points (p < 0.05 vs sham for the 1, 3, and 24 h post-reperfusion groups). Additionally, the hemoglobin volume at 3 and 24 h after reperfusion was significantly higher than the 15 min time point (p < 0.05 vs 15 min post-reperfusion for the 3 h, and 24 h post-reperfusion groups) (Fig. 2b).

Discussion

Hemorrhagic transformation occurs in a large number of ischemic stroke patients and contributes to the morbidity and mortality after stroke [11]. Several risk factors have been identified for causing hemorrhagic transformation, including tPA [19], hyperglycemia [9], and hypertension [9]. Clinically, treatment of hemorrhagic transformation relies primarily on surgical interventions, which may be ineffective [18]. Furthermore, pharmacologic interventions of hemorrhagic transformation are almost non-existent [1, 15, 18]. Thus it is critically important to develop novel therapeutics which can prevent the occurrence of, as well as treat, hemorrhagic transformation.

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