Spontaneously hypertensive rats display reduced microglial activation in response to ischemic stroke and lipopolysaccharide

Abstract

Background: For successful translation to clinical stroke studies, the Stroke Therapy Academic Industry Round Table criteria have been proposed. Two important criteria are testing of therapeutic interventions in conscious animals and the presence of a co-morbidity factor. We chose to work with hypertensive rats since hypertension is an important modifiable risk factor for stroke and influences the clinical outcome. We aimed to compare the susceptibility to ischemia in hypertensive rats with those in normotensive controls in a rat model for induction of ischemic stroke in conscious animals.

Methods: The vasoconstrictor endothelin-1 was stereotactically applied in the vicinity of the middle cerebral artery of control Wistar Kyoto rats (WKYRs) and spontaneously hypertensive rats (SHRs) to induce a transient decrease in striatal blood flow, which was measured by the laser Doppler technique. Infarct size was assessed histologically by cresyl violet staining. Sensory-motor functions were measured at several time points using the neurological deficit score. Activation of microglia and astrocytes in the striatum and cortex was investigated by immunohistochemistry using antibodies against CD68/Iba-1 and glial fibrillary acidic protein.

Results and conclusions: The SHRs showed significantly larger infarct volumes and more pronounced sensory-motor deficits, compared to the WKYRs at 24 h after the insult. However, both differences disappeared between 24 and 72 h. In SHRs, microglia were less susceptible to activation by lipopolysaccharide and there was a reduced microglial activation after induction of ischemic stroke. These quantitative and qualitative differences may be relevant for studying the efficacy of new treatments for stroke in accordance to the Stroke Therapy Academic Industry Round Table criteria.

Figure 1

A: Differences in blood flow reduction in the striatum of anesthetized WKYRs and SHRs. Shown are differences in blood flow reduction in the striatum of anesthetized WKYRs and SHRs after administration of 400 pmol Et-1. There were no differences between the two strains. *Significantly different from baseline levels. Differences between SHR and WKY were not significant for all time points as assessed by the unpaired Student’s t-test. (n = 4 for every group). B: Fixed fields in the striatum and cortex used for assessment of CD68 and GFAP expression. The three fixed fields are indicated in the striatum. Each has an area of 1 mm². Cells are counted in the three separate fields and the results are expressed as mean number of cells/mm². For the cortex, cells are counted between the reference lines. To determine the level of GFAP expression, the relative intensity is measured in the whole striatum and in the cortical region between the reference lines.

Figure 2

Differences in infarct volume between WKYRs and SHRs. Representative micrographs (scale bar 1 cm) of infarcts in a WKYR and SHR are shown in A and B, respectively. Brain slices were stained with Cresyl Violet 24 h after induction of the insult with 200 pmol Et-1. Graph C shows the differences in infarct volume between WKYRs and SHRs assessed after induction of focal cerebral ischemia with 180 or 200 pmol Et-1. *Significant difference between WKYRs and SHRs. ns: no significant difference between WKYRs and SHRs.

Figure 3

Neurological Deficit Scores in sham operated rats and Et-1 treated WKYRs and SHRs. Rats were injected with 200 pmol Et-1 and the NDS was assessed at different time points. At 24 h, there was a significant difference between the NDS of the two strains.. *Significant difference between WKYRs and SHRs. ^$ Significant difference between sham and Et-1 treated animals. ns: no significant difference between WKYRs and SHRs. Group sizes were n = 6 to 13 at 1 h, n = 5 to 10 at 6 h, n = 13 to 15 at 24 h, n = 3 to 5 at 48 h and n = 4 to 6 at 72 h.

Figure 4

Levels of CD68 positive cells in WKYRs and SHRs after Et-1 injection. Brain slices from WKYRs (n = 4) and SHRs (n = 4) treated with 200 pmol Et-1 were stained with anti-CD68 and the density of CD68⁺ cells was assessed in three fixed regions of the striatum and one region in the cortex (see Figure 1B) . Representative micrographs from two fixed regions in the striatum (A and D, upper left region; B and E upper right region) and one region in the cortex (C and F, in the center of the indicated region) are shown for WKYRs (A-C) and SHRs (D-F). Scale bar = 50 μm. Densities of CD68⁺ cells in the striatum (G) and cortex (H) are provided for both strains. The expression of CD68 remained low in the sham operated groups (n = 3 to 4 for the WKYR’s and n = 4 for the SHRs). *Significant difference between WKYRs and SHRs. ^$ Significant difference between sham and Et-1 treated animals. ns: no significant difference between WKYRs and SHRs.

Figure 5

GFAP expression levels in WKYRs and SHRs after Et-1 injection. Comparison of GFAP expression levels in the striatum (A) and cortex (B) of WKYRs and SHRs after induction of focal cerebral ischemia with 200 pmol Et-1. There is no difference between the two rats at every time point (n = 3 to 4 for the sham WKYRs and n = 4 to 5 for the Et-1 treated WKYRs and n = 3 to 4 for the sham SHRs and n = 4 to 5 for the Et-1 treated SHRs). *Significant difference between WKYRs and SHRs. ^$ Significant difference between sham and Et-1 treated animals. ns: no significant difference between WKYRs and SHRs.

Figure 6

CD68 (A-C) and Iba-1 expression levels (D-H) in WKYRs and SHRs 24 hrs after injection of LPS. The expression of CD68 is shown in a micrograph taken from a representative region in the striatum from WKYRs (A) and SHRs (B) and quantitative results are presented in the graph C. (The number of animals per group were n = 3 for the sham WKYRs and n = 4 for the sham SHRs; n = 4 for the LPS-treated groups). The expression of Iba-1 is shown for the ipsilateral (D, E) and contralateral hemispheres (F, G) for WKYRs (D, F) and SHRs (E, G). Graph H depicts quantitative differences in Iba-1 expression in the striatum of WKYRs and SHRs at 24 h after injection of 2 μg LPS (n = 4 for the all groups). *Significant difference between WKYRs and SHRs. ^$ Significant difference between sham and Et-1 treated animals. ns, no significant difference between WKYRs and SHRs. Scale bar A, B = 50 μm; scale bar D-G = 50 μm, scale bar insert = 20 μm.

Figure 7

iNOS expression levels in WKYRs and SHRs 24 hrs after injection of 2 μg LPS. Brains slices from WKYRs (A) and SHRs (B) were stained with an anti-iNOS antibody. C (WKY) and D (SHR) represent similar micrographs counterstained with Cresyl Violet and taken at a larger magnification. Cells expressing iNOS are indicated by arrows (C) or arrow heads (neurons in 7D). Scale bar = 20 μm.

Figure 8

GFAP expression levels in WKYRs and SHRs 24 h after injection of LPS. Comparison of GFAP expression levels in the striatum of WKYRs and SHRs after injection of 2, 4 and 40 μg LPS in the striatum (n = 3 for the sham WKY rats and n = 4 to 5 for the Et-1-treated WKY rats and n = 4 for the sham SHRs and n = 4 for the Et-1 treated SHRs). * Significant difference between WKY and SHR. ^$ Significant difference between sham and Et-1 treated animals. ns: no significant difference between WKY rats and SHRs.