Stroke induces histamine accumulation and mast cell degranulation in the neonatal rat brain

Abstract

Inflammatory processes are a major cause of hypoxic-ischemic brain damage. The present study focuses on both the cerebral histamine system and mast cells in a model of transient focal ischemia induced by permanent left middle cerebral artery, and homolateral transient common carotid artery occlusion (50 minutes) in the P7 newborn rat. Immunohistochemical analysis revealed that ischemia induces histamine (HA) accumulation in the core of the infarct 6-12 h post-ischemia, and in the penumbra at 24-48 h, although in situ hybridization failed to detect any histidine decarboxylase gene transcripts in these regions. Immunohistochemical co-localization of HA with the MAP2 marker revealed that HA accumulates in neuronal cells before they degenerate, and is accompanied by a very significant increase in the number of mast cells at 12 h and 48 h of reperfusion. In mast cells, histamine immunoreactivity is detected at 2, 6 and 12 h after ischemia, whereas it disappears at 24 h, when a concomitant degranulation of mast cells is observed. Taken together, these data suggest that the recruitment of cerebral mast cells releasing histamine may contribute to ischemia-induced neuronal death in the immature brain.

Figure 1

Specificity of the antihistamine immunostaining. Ischemic brain sections were incubated with a polyclonal antihistamine antibody in the absence (A) or presence (B,C) of histamine‐ovalbumine at 10 µg/ml (B) and 100 µg/ml (C). Note that the two concentrations of histamine completely abolished the immunolabeling (core of the infarct, 24 h after ischemia). Scale bar = 50 µm.

Figure 2

Histopathological changes of the left cerebral hemisphere after neonatal ischemia in P7 rats. A. Representative cresyl‐violet‐stained coronal brain section, 48 h following ischemia. Note the large ill‐defined pale area corresponding to the infarct (core C; and penumbra P). B,C. Ischemia‐induced cell death in the parietal cortex evidenced by DNA fragmentation and apoptotic bodies using fluorescent TUNEL assay in the core (B) and penumbra (C). Scale bar = 25 µm.

Figure 3

Histamine accumulation in the cerebral cortex after neonatal stroke in the P7 rat. Ischemia induced a cortical infarct as shown in the diagram (C, core; P, penumbra). A–D. Histamine‐like immunoreactivity (HA‐like IR) observed in the core of the infarct. A. 2 h after ischemia, HA‐like IR transiently appeared in cortical blood vessels (V) of the ischemic core. B. At 6 h post ischemia, a faint immunostaining occurred in pyramidal‐shaped cortical cells (arrows). C. 24 h after reperfusion, HA‐like IR increased in intensity and was observed within many cells of the core. D. At 48 h, HA‐like IR phagocytic cells (arrow heads) were observed in the superficial cortical layers; blood vessels (v) were not labeled. E. Cortical cells not subjected to ischemia did not show any HA‐like IR. F,G. HA‐like IR in the penumbra at 24 h (F) and 48 h (G) post ischemia. H. HA‐like IR in histaminergic magnocellular neurons of the tuberomammillary nucleus of the hypothalamus. I–K. In situ hybridization of histidine decarboxylase (hdc) gene transcripts did not reveal any signal in the ischemic infarct, neither 6 h (I), nor 24 h (J), following ischemia. K. A mast cell of the pia matter expressing hdc mRNAs. In A,C,D,E,G,I,J, brain tissues were counterstained with hemalun to vizualize cell nuclei. Photographs are representative of three different experiments on each animal. Scale bars represent 25 µm (A,D,F,H), 50 µm (B,C,E,G), 100 µm (I,J) and 10 µm (K).

Figure 4

Co‐localization of histamine and the neuronal marker MAP2, 48 h after neonatal stroke, in the ipsilateral cortex at the limit core/penumbra (A–E) and in a non‐ischemic zone (F–I). The fluorescent labeling was evidenced with antibodies against histamine (red, A,F), MAP2 (green, B,G), and nuclear marker Hoechst 33342 (blue, D,H). Co‐localization of HA and MAP2 is shown in C and co‐localization of the three markers is shown in E and I. In the ischemic cortex (A–E), labeling demonstrates the co‐localization of histamine‐like immunoreactivity (IR) with MAP2 IR in the penumbra (C,E): in degenerating neurons at the beginning of the degenerative process (double arrow), at the end of dendritic loss (arrow head) and in neurons with edematous aspect (*). Note that in the degenerating neurons, MAP2 IR resulted in segmental dendritic beading or varicosities and loss of dendrites (B). In the core, Histamine‐like IR and MAP2 IR disappeared and neurons presented apoptotic characteristics with condensed chromatin and the presence of apoptotic bodies (arrows, E). In the control zone of the cortex (F–I), Histamine‐like IR was not detected (F) and MAP2 staining revealed dendritic processes and neuronal cell bodies characteristic of healthy neurons (G,I). V: blood vessel. Scale bar = 15 µm.

Figure 5

Mast cells of the choroïd fissure after neonatal stroke. A–F. Mast cells containing histamine immunostaining were observed as early as 2 h (A), 6 h (B,E), 12 h (C) and 24 h (D,F) after ischemia. The marked decrease in histamine immunolabeling observed at 24 h (D,F) suggests that mast cells have degranulated. G–K. Acidic toluidine blue staining of mast cells in P7 control (I) and ischemic (J,K) rat brains. Note the presence at 24 h, of a great number of degranulating mast cells (K), at different states of mast cell degranulation: (G) mast cells poorly degranulated, (H) mast cell highly degranulated. Scale bars represent 40 µm (A–D,I–K), 10 µm (E,F) and 20 µm (G,H).