Activation of the complement cascade and increase of clusterin in the brain following a cortical contusion in the adult rat

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✓ The aim of the present study was to examine the glial cell response and the possible involvement of the complement cascade following a cerebral cortical contusion. The lesion was produced using a standardized weight-drop technique in adult rats. The blood-brain barrier was damaged, as demonstrated by a decrease of immunoreactivity for a tight junction protein normally expressed by endothelial cells of small vessels in the central nervous system. Increased immunoreactivity for microglial (OX42) and astroglial cells (glial fibrillary acidic protein), as well as macrophages expressing ED1-immunoreactivity (IR) were found in the vicinity of the lesion at all postoperative survival times (2–14 days).

In the present study complement factor C3d- and C9-IR was found around the lesion, indicating that activation of the complement cascade had taken place. Furthermore, immunoreactivity for the putative complement inhibitor clusterin (sulfated glycoprotein-2) was found in some of the injured neurons. The contralateral hemisphere showed no evidence of the reaction found in the ipsilateral hemisphere. The balance between complement activation and complement inhibitors may have an impact on the degenerative components in the brain following traumatic injury and in particular on the events leading to nerve cell death.

Article Information

Address reprint requests to: Mikael Svensson, M.D., Ph.D., Karolinska Institutet, Department of Neuroscience, Doktorsringen 17 (Anatomy Building), S-171 77 Stockholm, Sweden.

© AANS, except where prohibited by US copyright law.

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Figures

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    Simplified schematic illustration showing the complement cascade. The complement factors are present normally in plasma or synthesized locally in the tissue by macrophages. These proteins may become activated via the classic pathway by immunoglobulins or via the alternative pathway. Fragments of C3 and C5 stimulate recruitment of macrophages (chemotaxis) and facilitate phagocytosis (opsonization). The endproduct C5b-9, membrane attack complex, may cause lysis of target cells. IgG = immunoglobulin G.

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    Photomontage showing OX42-immunoreactivity (IR) in intact and injured rat brains. a: Photomicrograph of a frontally cut section of the brain showing OX42-IR 7 days after the contusion. Note increased immunoreactivity on the injured side (arrow). b: The injured side at higher magnification showing activated microglial cells with thick processes. c: The uninjured side showing resting microglial cells with numerous thin processes. d: Glial fibrillary acidic protein—IR in the contusion area, 7 days postlesioning. Note the intense labeling in hypertrophied astrocytes. e: Normal astrocytes on the contralateral side with slender processes. Bars = 100 µm (a), 10 µm (b and c), and 50 µm (d and e).

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    Photomontage showing immunoreactivity for a tight junction protein in intact and injured rat brains. a: Photomicrograph of the injured side showing immunoreactivity for a tight junction protein 4 days postlesioning. Note the absence of immunoreactivity in the vessels surrounding the contusion, reflecting the damage to the blood-brain barrier. b: Higher magnification of an area adjacent to the contusion showing vessels with low or no immunoreactivity. c: Control side. Note vessels expressing tight junction-immunoreactivity (IR). d and e: Photomicrographs showing presence or absence of ED1-IR 4 days after injury. Note the great number of ED1-positive cells in the contusion (d) and in higher magnification (inset). No ED1-IR was found on the uninjured side (e). Bars = 300 µm (a), 50 µm (b and c), 300 µm (d and e), and 100 µm (inset).

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    Photomicrographs. a and b: Complement component C3d- (a) and C9-immunoreactivity (IR) (b), 4 days postinjury. The immunoreactivity is present in neurons and in the neuropil in the vicinity of the contusion, which is shown in higher magnifications in c to f. c and d: Immunoreactivity to C3d-IR, 4 days postinjury. Note C3d-IR (c, injured side) in a granular pattern (arrows) or in close to the cell membranes of neuronal perikarya (asterisks). Uninjured side (d). e and f: Immunoreactivity to C9-IR 4 days postlesioning. Injured side (e). Note the intense immunoreactivity in presumed degenerative neuronal perikarya (asterisks) located in the contusion area as well as granular labeling in the neuropil (arrows). Uninjured side (f). Bars = 200 µm (a and b), and 10 µm (c–f).

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    Photomicrographs showing clusterin-immunoreactivity 7 days postinjury (a). Note the increase of immunoreactivity in neuronal perikarya in comparison to the uninjured side (b). Bar = 50 µm.

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    Photomicrographs showing double labelings (arrows) 4 days postinjury. Neurons in the vicinity of the contusion were double stained with anti-MAP5 (a) and anticlusterin (b). Note the overlap in immunoreactivity. Astroglial cells double labeled with antiglial fibrillary acidic protein (c) and anticlusterin (d). Reactive microglia double stained with OX42 (e) and anti-C3 (f). Fluorescein isothiocyanate (a, c, and e) and rhodamine (b, d, and f). Bar = 50 µm.

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    a: Autoradiogram showing glial fibrillary acidic protein—messenger RNA 7 days postlesioning. Note the intense reaction in the contusion area (arrow). b: Control probe. c: Injured side. Note the intense grain density over counterstained astroglial nuclei. d: Darkfield micrograph from the same area as in c. e: Uninjured side. Note the relatively low level of grain density in comparison to the injured side. f: Darkfield micrograph from the same area as in e. Bars = 2 mm (a) and 50 µm (b–f).

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    a: Autoradiogram showing C3—messenger RNA, 4 days postlesioning. Note the intense reaction in the contusion area (arrow). The border of the section has been indicated with a white line. b: Control probe. c: Injured side with intense grain density over counterstained nuclei representing reactive microglia and most likely also other macrophages derived from the blood. d: Darkfield micrograph from the same area as in c. e: Uninjured side. f: Darkfield micrograph from the same area as in e. Bars = 2 mm (a) and 50 µm (b–f).

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    a: Autoradiogram showing clusterin—messenger RNA 7 days postlesioning. Note the intense reaction in the contusion area (arrow). b: Control probe. c: Injured side. Note the intense grain density over some of the counterstained neuronal perikarya (arrows). However, some neurons in the same area showed low grain density (arrowheads). d: Darkfield micrograph from the same area as in c. e: Uninjured side. Note the low density of grains. f: Darkfield micrograph from the same area as in e. Bars = 2 mm (a) and 50 µm (b–f).

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