Astrocytosis, Inflammation, Axonal Damage and Myelin Impairment in the Internal Capsule following Striatal Ischemic Injury

Abstract

Secondary degeneration is defined as a set of destructive events that damage cells and structures that were initially spared or only peripherally affected by the primary insult, constituting a key factor for functional impairment after traumatic brain injury or stroke. In the present study, we evaluated the patterns of astrocytosis, inflammatory response, axonal damage and oligodendrocytes/myelin impairment in the internal capsule following a focal injection of endothelin-1 (ET-1) into the dorsal striatum. Animals were perfused at 1, 3 and 7 post-lesion days (PLD), and tissue was processed to immunohistochemistry for neutrophils (MBS1), macrophages/microglia (ED1), astrocytes (GFAP), axonal lesion (βAPP), oligodendrocytes (Tau) and myelin (MBP). A significant number of neutrophils was observed at 1PLD, followed by intense recruitment/activation of macrophages/microglia at 3PLD and astrocytic reaction with a peak at 7PLD. Oligodendrocyte damage was pronounced at 3PLD, remaining at 7PLD. Progressive myelin impairment was observed, with reduction of immunoreactivity at 7PLD. Axonal lesion was also identified, mainly at 7PLD. Our results indicate that acute inflammatory response elicited by the ischemic insult in the striatum can be associated with the axonal impairment and damage of both oligodendrocytes and myelin sheath identified in the internal capsule, which may be related to loss of tissue functionality observed in secondary degeneration.

Keywords: inflammation; internal capsule; rat; secondary degeneration; stroke.

Figure 1

Schematic figure summarizing the methodological design. The animals were placed in a stereotaxic apparatus and a focal ischemia was induced by injection of endothelin-1 (ET-1) into the dorsal striatum. After the specified post-lesion times, animals were perfused and their brains sectioned and processed for being evaluated under bright-field microscopy.

Figure 2

Basic histology (Nissl staining) following striatal endothelin-1 (ET-1) injection. There was a loss of tissue after seven post-lesion days (7PLD) (A), identified by a well-defined region of pallor, not observed in the control animal (B). Dashed squares in lower magnifications point to the location where higher power was obtained. Arrowheads point to the center of the injection. Drawings at the left side of the figure show the anatomical location of the ET-1 injection site into the striatum. Ctx: cortex; CPu: caudate putamen (striatum); Di: diencephalon; Hip: hippocampus; ic: internal capsule. Scale bars: 2 mm (lower magnification); 200 μm (enlargements).

Figure 3

Neutrophil recruitment in the internal capsule following endothelin-1 (ET-1) injection. While in control group the inflammatory response is negligible (A), an intense recruitment of neutrophils is observed one day after injection (1PLD) (B), with a significant decrease at 3PLD (C). Neutrophil response is absent at 7PLD (D), as confirmed by quantitative analysis (E) (** p < 0.01; *** p < 0.001, ANOVA, Tukey post hoc test). Values expressed as mean ± SEM. Arrows in A and B point to MBS1⁺ neutrophils. Drawings at the left side of the figure show the anatomical localization of the ET-1 injection in the striatum. Ctx: cortex; CPu: caudate putamen (striatum); Di: diencephalon; Hip: hippocampus; ic: internal capsule. Scale bar: 50 µm.

Figure 4

Progressive macrophage/microglia activation in the internal capsule following endothelin-1 (ET-1) striatal injection. The inflammatory response is minimal in the control group (A). At 1PLD it is possible to notice a small number of ED1⁺ cells (B), followed by a significant increase at 3PLD (C), with a decrease at 7PLD (D), as confirmed by quantitative analysis (E) (* p < 0.05; ** p < 0.01; *** p < 0.001, ANOVA, Tukey post hoc test). Values expressed as mean ± SEM. Arrows in B, C and D point to ED1⁺ cells. Drawings at the left side of the figure show the anatomical localization of the ET-1 injection in the striatum. Ctx: cortex; CPu: caudate putamen (striatum); Di: diencephalon; Hip: hippocampus; ic: internal capsule. Scale bar: 50 µm.

Figure 5

Astrocytosis in the internal capsule following endothelin-1 (ET-1) striatal injection. Non-activated astrocytes are observed in control group (A). A progressive astrocytosis is seen at 1PLD (B) and 3PLD (C), with hypertrophic cell bodies and short and thick processes being observed at 7PLD (D). Drawings at the left side of the figure show the anatomical localization of the ET-1 injection in the striatum. Ctx: cortex; CPu: caudate putamen (striatum); Di: diencephalon; Hip: hippocampus; ic: internal capsule. Scale bar: 50 µm.

Figure 6

Oligodendrocyte damage in the internal capsule following endothelin-1 (ET-1) injection. Control (A) and endothelin-1-injected groups at 1PLD (B), 3PLD (C) and 7PLD (D). The peak of Tau⁺ cells is observed at 3PLD, as confirmed by quantitative analysis (E) (* p < 0.05; *** p < 0.001, ANOVA, Tukey post hoc test). Values expressed as mean ± SEM. Arrows in (C,D) point to TAU⁺ oligodendrocytes. Drawings at the left side of the figure show the anatomical localization of the ET-1 injection in the striatum. Ctx: cortex; CPu: caudate putamen (striatum); Di: diencephalon; Hip: hippocampus; ic: internal capsule. Scale bar: 50 µm.

Figure 7

Apoptotic profiles following oligodendrocyte lesion in the internal capsule induced by endothelin-1 (ET-1) striatal injection. Nissl counterstaining revealed the presence of pycnotic bodies (arrowheads), a result confirmed by immunohistochemistry for caspase-3 (arrows) (B). Arrows in (A) point to Tau⁺ oligodendrocytes. Drawings at the left side of the figure show the anatomical localization of the ET-1 injection in the striatum. Ctx: cortex; CPu: caudate putamen (striatum); Di: diencephalon; Hip: hippocampus; ic: internal capsule. Scale bar: 100 µm.

Figure 8

Axonal lesion in the internal capsule following endothelin-1 (ET-1) striatal injection. Control (A) and ET-1-injected groups at 1PLD (B), 3PLD (C) and 7PLD (D). The peak of APP labeling is observed at 7PLD, evidenced by a diffuse axon labeling (arrows in (D)). Caspase-3 immunohistochemistry allowing identifying a similar pattern of immunoreactivity, with intense labeling at 7PLD (F) (arrows). Control group presenting the absence of caspase-3 labeling (E). Progressive APP labeling across time points was confirmed by densitometric analysis (G) (* p < 0.05; ** p < 0.01, ANOVA, Tukey post hoc test). Drawings at the left side of the figure show the anatomical localization of the ET-1 injection in the striatum. Ctx: cortex; CPu: caudate putamen (striatum); Di: diencephalon; Hip: hippocampus; ic: internal capsule. Scale bar: 50 µm.

Figure 9

Myelin impairment in the internal capsule following endothelin-1 (ET-1) injection. Control group shows a homogeneous MBP⁺ labeling (A). A progressive impairment is observed at 1PLD (B), 3PLD (C) and 7PLD (D), with a noticeable rarefaction of reactivity in the latter survival time. Vacuolization, a hallmark of tissue degeneration, is also evident at 7PLD (arrows). Progressive loss of MBP labeling across time points was confirmed by densitometric analysis (E) (* p < 0.05; ** p < 0.01, ANOVA, Tukey post hoc test). Drawings at the left side of the figure show the anatomical localization of the ET-1 injection in the striatum. Ctx: cortex; CPu: caudate putamen (striatum); Di: diencephalon; Hip: hippocampus; ic: internal capsule. Scale bar: 100 µm.