Early treatment with minocycline following stroke in rats improves functional recovery and differentially modifies responses of peri-infarct microglia and astrocytes

Abstract

Background: Altered neuronal connectivity in peri-infarct tissue is an important contributor to both the spontaneous recovery of neurological function that commonly develops after stroke and improvements in recovery that have been induced by experimental treatments in animal models. Microglia and astrocytes are primary determinants of the environment in peri-infarct tissue and hence strongly influence the potential for neuronal plasticity. However, the specific roles of these cells and the timing of critical changes in their function are not well understood. Minocycline can protect against ischemic damage and promote recovery. These effects are usually attributed, at least partially, to the ability of this drug to suppress microglial activation. This study tested the ability of minocycline treatment early after stroke to modify reactive responses in microglia and astrocytes and improve recovery.

Methods: Stroke was induced by photothrombosis in the forelimb sensorimotor cortex of Sprague-Dawley rats. Minocycline was administered for 2 days after stroke induction and the effects on forelimb function assessed up to 28 days. The responses of peri-infarct Iba1-positive cells and astrocytes were evaluated using immunohistochemistry and Western blots.

Results: Initial characterization showed that the numbers of Iba1-positive microglia and macrophages decreased in peri-infarct tissue at 24 h then increased markedly over the next few days. Morphological changes characteristic of activation were readily apparent by 3 h and increased by 24 h. Minocycline treatment improved the rate of recovery of motor function as measured by a forelimb placing test but did not alter infarct volume. At 3 days, there were only minor effects on core features of peri-infarct microglial reactivity including the morphological changes and increased density of Iba1-positive cells. The treatment caused a decrease of 57% in the small subpopulation of cells that expressed CD68, a marker of phagocytosis. At 7 days, the expression of glial fibrillary acidic protein and vimentin was markedly increased by minocycline treatment, indicating enhanced reactive astrogliosis.

Conclusions: Early post-stroke treatment with minocycline improved recovery but had little effect on key features of microglial activation. Both the decrease in CD68-positive cells and the increased activation of astrogliosis could influence neuronal plasticity and contribute to the improved recovery.

Keywords: Astrocytes; Focal ischemia; Functional recovery; Microglia; Minocycline; Peri-infarct; Photothrombosis; Stroke.

Conflict of interest statement

Ethics approval and consent to participate

All studies involving rats were conducted according to the “Australian code for the care and use of animals for scientific purposes, 2013” published by the National Health and Medical Research Council, Australia. The investigations were approved by the Animal Welfare Committee of Flinders University, Adelaide, South Australia (Project number 754/10).

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

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Figures

Fig. 1

Summary of experimental design for the three major phases of the study. a Characterization of responses of Iba1-positive cells in peri-infarct tissue during the first week following photothrombotic stroke. The numbers of rats shown were all assessed using the forelimb placing test. Perfusion-fixed tissue from four to six rats per time point was used to assess infarct volume and that from four rats per time point was used for immunohistochemistry. b Effects of lower-dose minocycline treatment on forelimb function and cellular changes during the first week following photothrombotic stroke. c Effects of higher-dose minocycline treatment on forelimb function (up to 28 days) and cellular changes following photothrombotic stroke

Fig. 2

Tissue damage and functional changes following photothrombotic stroke. a Cresyl-violet stained coronal section of a rat brain at 24 h after induction of photothrombotic stroke showing the clearly defined infarct and the ROIs used for analysis of sections immunolabeled to detect Iba1. The scale is in millimeters. b Cresyl-violet stained coronal section at 3 h after stroke induction. c Infarct volume over 7 days after stroke induction. The infarct is fully developed by 24 h and then contracts substantially by 7 days (n = 4–6 per time point). d Coronal section of cerebral cortex immunolabeled for NeuN at 3 h after stroke induction (scale bar = 500 μm). e Box plot of forelimb placing scores following stroke. Outlier values are shown as circles; Blue = forelimb ipsilateral to infarct, Red = forelimb contralateral to infarct (n ≥ 5 per group)

Fig. 3

Circularity of Iba1-positive cells after photothrombotic stroke. This parameter was measured in ROIs as defined in Fig. 2a. a Comparison of Iba1-immunolabeled cells in peri-infarct tissue (Ipsi01A) and equivalent tissue (Contra01) in the hemisphere contralateral to the infarct at 3 h after stroke induction. The scale bar = 100 μm. b Regional differences at 3 h. Insets show examples of processed images of Iba1-positive cells at an ROI adjacent to the infarct (Ipsi01A) and in corresponding contralateral tissue (Contra01). c Regional differences at 24 h. Inset shows an example of a processed image at Ipsi01A. d Time course of changes in peri-infarct regions (Ipsi01A and Ipsi01B), tissue 1 mm from the infarct (ipsi01A + 1 mm) and corresponding contralateral tissue (Contra01) over the first 7 days. For panels b–d, n = 4 for each data point; *p < 0.05, **p < 0.01, ***p < 0.001 compared with the corresponding contralateral region (one-way ANOVA with Tukey’s HSD post-hoc test)

Fig. 4

Additional morphological features of Iba1-positive cells at 24 h and 7 days after photothrombotic stroke. a Cell area, b cell perimeter, and c cell soma area. For panels a–c, n = 3 (7 days) or 4 (24 h); *p < 0.05, **p < 0.01, ***p < 0.001 compared with the corresponding contralateral region, contra01 (one-way ANOVA with Tukey’s HSD post-hoc test). The ROIs that were analyzed are identified in Fig. 2a

Fig. 5

Changes in the pattern of Iba1-immunolabeling after photothrombotic stroke. a Regional differences in area fraction at 24 h. b–d Changes in b area fraction, c Iba1-positive particle count, and d Iba1-positive particle size in peri-infarct tissue, tissue 1 mm from the infarct and corresponding contralateral tissue between 3 h and 7 days. For all panels, n = 4 per data point; *p < 0.05, **p < 0.01, ***p < 0.001 compared with Contra01 (one-way ANOVA with Tukey’s HSD post-hoc test). At 24 h, there was also a significant difference between each of the peri-infarct regions and tissue located 1 mm from the infarct (at ipsi01a + 1 mm) for area fraction (Ipsi01a: p < 0.05; Ipsi01b: p < 0.01) and particle count (p < 0.01 for each of the peri-infarct regions). The ROIs that were analyzed are identified in Fig. 2a

Fig. 6

Effects of lower dose minocycline treatment on infarct volume and forelimb function after photothrombotic stroke. a Infarct volume: Two-way analysis of variance revealed no significant effect of the minocycline treatment but a significant effect of time after stroke (p < 0.01; n = 4–8 per group). b Box plot of results from the forelimb placing test. Outliers are indicated with open circles. n = 6 per group for all times except 3 h which shows the pooled data for rats in each of the subsequent groups. Rate of recovery was faster at 3 days *p < 0.05 (Minocycline vs vehicle, Mann-Whitney U test analysis of area under curve for those rats tested at both 3 h and 3 days). No significant differences were detected at other times

Fig. 7

Effects of lower dose minocycline treatment on responses of Iba1-positive cells after photothrombotic stroke. a Circularity and b area fraction of Iba1-positive cells at 24 h, 3 days and 7 days following photothrombotic stroke (n = 4 to 8 per group). Two-way analysis of variance detected highly significant differences between the contralateral and peri-infarct regions for both circularity and area fraction at all three time points (p < 0.001). However, there were no significant effects of treatment on these parameters

Fig. 8

Effect of higher-dose minocycline treatment on recovery of neurological function after photothrombotic stroke. Box plots of dysfunction and recovery up to 28 days after photothrombotic stroke in a the forelimb placing test and b the cylinder test. Outliers are shown as open circles (n = 8 per group). Recovery in the forelimb placing test was more rapid in minocycline-treated rats (p < 0.05; Mann-Whitney U test comparison of area under curve for time points between 3 h and 28 days.) There was no significant difference in recovery between 24 h and 28 days for performance in the cylinder test

Fig. 9

Effect of higher-dose minocycline treatment on Iba1-positive cells at 3 days after photothrombotic stroke. a Circularity and b area fraction of immunolabeling (n = 6 to 7 per group). Two-way analysis of variance revealed a statistically significant difference between the hemispheres for both circularity and area fraction (p < 0.01) and a significant effect of the minocycline treatment on Iba1 area fraction (p < 0.05) but not on circularity. For area fraction, there was no significant interaction between hemisphere and minocycline treatment

Fig. 10

Effect of higher-dose minocycline treatment on CD68-immunolabeled cells at 3 days after photothrombotic stroke. a Representative image of CD68 (red) and NeuN (green) double-immunolabeled coronal section at the infarct boundary of a vehicle-treated rat. The infarct is visible in the upper part of the image. The scale bar = 200 μm. b Effects of the higher-dose minocycline treatment on CD68-positive cells within the peri-infarct tissue at 3 days following photothrombotic stroke (n = 6 to 7 per group). The CD68-positive particle count was assessed within 0.3 mm of the infarct (Peri-infarct) and between 0.3 and 0.6 mm from the infarct (Peri-infarct + 0.3 mm). Two-way analysis of variance revealed a highly significant effect of both treatment (p < 0.01) and tissue location (p < 0.001) on CD68-positive particle count at 3 days after stroke. There was also a strong interaction between the two factors (p < 0.001) reflecting the larger decrease in particle density induced by minocycline in the tissue immediately adjacent to the infarct compared with the other regions examined

Fig. 11

Effects of higher-dose minocycline treatment on markers of reactive astrogliosis after photothrombotic stroke. a, b A large increase in vimentin content in peri-infarct tissue over the first 7 days following photothrombotic stroke in untreated rats was detected by a area fraction of vimentin-positive cells and b Western blotting. The area fraction of vimentin immunolabeling was markedly increased by 2 days and further increased at 3 and 7 days after stroke (n = 4 per group at each time); *p < 0.05, **p < 0.01 vs. corresponding contralateral region (one-way ANOVA with Tukey’s HSD). c Double immunolabeling of GFAP and vimentin in peri-infarct tissue at 3 days after photothrombotic stroke. Vimentin (red) is expressed almost exclusively in cells also expressing GFAP (green). Yellow shows colocalization. The scale bar = 100 μm. d–e Effect of higher-dose minocycline treatment on expression of astroglial proteins in samples containing infarct plus peri-infarct tissue at 7 days following photothrombotic stroke (n = 6 per group). d The intermediate filament proteins, vimentin and GFAP. e Neurocan. The band intensities for vimentin (54 kDa) and GFAP (50 kDa) were normalized to actin (45 kDa). There was a significant effect of minocycline treatment on both vimentin and GFAP content (**p < 0.01). The intensity of bands for full-length neurocan (approx. 250 kDa) were determined relative to that of a proteolytic fragment of this protein (approx. 150 kDa). No statistically significant effects were detected in the neurocan protein ratio in tissue from the minocycline-treated rats compared with the vehicle-treated rats