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. 2012 May 9;56(2):e14.
doi: 10.4081/ejh.2012.14.

Spatio-temporal course of macrophage-like cell accumulation after experimental embolic stroke depending on treatment with tissue plasminogen activator and its combination with hyperbaric oxygenation

D Michalski  1 , M Heindl, J Kacza, F Laignel, L Küppers-Tiedt, D Schneider, J Grosche, J Boltze, M Löhr, C Hobohm, W Härtig
Affiliations

Spatio-temporal course of macrophage-like cell accumulation after experimental embolic stroke depending on treatment with tissue plasminogen activator and its combination with hyperbaric oxygenation

D Michalski et al. Eur J Histochem. .

Abstract

Inflammation following ischaemic stroke attracts high priority in current research, particularly using human-like models and long-term observation periods considering translational aspects. The present study aimed on the spatio-temporal course of macrophage-like cell accumulation after experimental thromboembolic stroke and addressed microglial and astroglial reactions in the ischaemic border zone. Further, effects of tissue plasminogen activator (tPA) as currently best treatment for stroke and the potentially neuroprotective co-administration of hyperbaric oxygen (HBO) were investigated. Rats underwent middle cerebral artery occlusion and were assigned to control, tPA or tPA+HBO. Twenty-four hours, 7, 14 and 28 days were determined as observation time points. The accumulation of macrophage-like cells was semiquantitatively assessed by CD68 staining in the ischaemic area and ischaemic border zone, and linked to the clinical course. CD11b, ionized calcium binding adaptor molecule 1 (Iba), glial fibrillary acidic protein (GFAP) and Neuronal Nuclei (NeuN) were applied to reveal delayed glial and neuronal alterations. In all groups, the accumulation of macrophage-like cells increased distinctly from 24 hours to 7 days post ischaemia. tPA+HBO tended to decrease macrophage-like cell accumulation at day 14 and 28. Overall, a trend towards an association of increased accumulation and pronounced reduction of the neurological deficit was found. Concerning delayed inflammatory reactions, an activation of microglia and astrocytes with co-occurring neuronal loss was observed on day 28. Thereby, astrogliosis was found circularly in contrast to microglial activation directly in the ischaemic area. This study supports previous data on long-lasting inflammatory processes following experimental stroke, and additionally provides region-specific details on glial reactions. The tendency towards a decreasing macrophage-like cell accumulation after tPA+HBO needs to be discussed critically since neuroprotective properties were recently ascribed to long-term inflammatory processes.

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Conflict of interest statement

Conflict of interest: the authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Figure 1. Overview scan (A) of a coronal brain section in a rat with focal cerebral ischaemia, located in the right middle cerebral artery territory affecting the thalamus, with accumulation of macrophage-like cells, revealed by immunoperoxidase staining of CD68. Scans with 25× magnification in the ischaemic area and in the ischaemic border zone – exemplarily shown in (B) and (C) – were used for semi-quantitative analysis of CD68-immunopositive cells. Scale bars: A), 1 mm; B–C), 50 µm.
Figure 2
Figure 2
Spatio-temporal course of pixel-based calculations per optic field detecting the accumulation of CD68-positive cell in two different ischaemia-related areas depending on different treatment regimes. Symbols represent means and added bars represent standards errors. Sample size: Three sections were analysed in each case, 3 subjects served for each treatment group at each time point.
Figure 3
Figure 3
Scatter plots indicating the interrelation between the pixel-based ratio of CD68-immunoreactivity (as surrogate for the accumulation of macrophage-like cells) and the clinical impairment (shown as course of the neurological deficit during the overall observation period). Thereby, increasing values of the CD68-positive pixel ratio represent an increasing accumulation and tended to be associated with clinical improvement. Beta and P-value originated from linear regression analysis using the pixel ratio as independent and the change of neurological deficit as dependent factor.
Figure 4
Figure 4
Triple fluorescence labelling of markers for macrophages and microglia, 4 weeks after embolic stroke in the right middle cerebral artery territory. (A) The staining of CD68 with FITC-conjugated monoclonal mouse antibodies was enhanced by Cy2-tagged anti-FITC, and combined with (B) the immunodetection of CD11b based on biotinylated mouse antibodies visualized by Cy3-streptavidin, and (C) Cy5-immunolabelling of ionized calcium binding adaptor molecule 1 (Iba) in microglial cells. The merge of the three staining patterns reveals a microglial population co-expressing CD11b and Iba (purple, arrows), but in the centre also cells exist, displaying all three markers (white, arrowheads). Scale bar: 50 µm.
Figure 5
Figure 5
Concomitant immunofluorescence staining of CD68 in macrophage-like cells, ionized calcium binding adaptor molecule 1 (Iba) in microglial cells and glial fibrillary acidic protein (GFAP) in astroglia 4 weeks after embolic stroke in the border zone of the infarct. (A) CD68-immunoreactivity addressed with biotinylated monoclonal mouse antibodies and visualised by Cy2-conjugated streptavidin, and (B) Iba revealed by Cy5-immunolabelling were counterstained in (C) with Cy3-coupled mouse-anti-GFAP. By merging of staining patterns in (D) activated micro- and astroglia were also elucidated around the centre of the infarct with infiltrated CD68-immunopositive macrophages. Scale bar: 50 µm.
Figure 6
Figure 6
Stroke-induced glial activation corresponding to neuronal loss in the neocortex. Four weeks after embolic stroke, the outer rim of the infarct zone in the left part of the pictures displays in (A) activated microglia revealed by Cy5-immunolabelling of ionized calcium binding adaptor molecule 1 (Iba) and in (B) a strong, infarct-induced astrogliosis shown by Cy3-staining of glial fibrillary acidic protein (GFAP). In (C), the Cy2-immunolabelling of the pan-neuronal marker NeuN reveals the loss of neuronal structures in the area of infarction. The overlay of staining patterns in (D) visualises the border between the stroke-affected region (displaying gliosis and neuronal loss) and the apparently healthy tissue (without apparent glial activation and non-affected neuronal staining). Scale bar: 100 µm.
Figure 7
Figure 7
Simultaneous detection of glia and neurons 4 weeks after embolic stroke in the hippocampal border zone. Activated microglia are shown in (A) by ionized calcium binding adaptor molecule 1 (Iba)-immunostaining (Cy5), whereas (B) demonstrates the astrogliosis revealed by Cy3-staining of glial fibrillary acidic protein (GFAP) and (C) displays a slightly diminished Cy2-staining of neuronal nuclei (NeuN) in the area of and surrounding the pyramidal layer of the CA1 region. By merging the staining patterns in (D) the stroke-induced gliosis becomes even clearer (in the upper right corner of the picture). Scale bar: 100 µm.
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