Formation of multinucleated giant cells and microglial degeneration in rats expressing a mutant Cu/Zn superoxide dismutase gene

Abstract

Background: Microglial neuroinflammation is thought to play a role in the pathogenesis of amyotrophic lateral sclerosis (ALS). The purpose of this study was to provide a histopathological evaluation of the microglial neuroinflammatory response in a rodent model of ALS, the SOD1G93A transgenic rat.

Methods: Multiple levels of the CNS from spinal cord to cerebral cortex were studied in SOD1G93A transgenic rats during three stages of natural disease progression, including presymptomatic, early symptomatic (onset), and late symptomatic (end stage), using immuno- and lectin histochemical markers for microglia, such as OX-42, OX-6, and Griffonia simplicifolia isolectin B4.

Results: Our studies revealed abnormal aggregates of microglia forming in the spinal cord as early as the presymptomatic stage. During the symptomatic stages there was prominent formation of multinucleated giant cells through fusion of microglial cells in the spinal cord, brainstem, and red nucleus of the midbrain. Other brain regions, including substantia nigra, cranial nerve nuclei, hippocampus and cortex showed normal appearing microglia. In animals during end stage disease at 4-5 months of age virtually all microglia in the spinal cord gray matter showed extensive fragmentation of their cytoplasm (cytorrhexis), indicative of widespread microglial degeneration. Few microglia exhibiting nuclear fragmentation (karyorrhexis) indicative of apoptosis were identified at any stage.

Conclusion: The current findings demonstrate the occurrence of severe abnormalities in microglia, such as cell fusions and cytorrhexis, which may be the result of expression of mutant SOD1 in these cells. The microglial changes observed are different from those that accompany normal microglial activation, and they demonstrate that aberrant activation and degeneration of microglia is part of the pathogenesis of motor neuron disease.

Figure 1

Microglial staining with OX-42 immunohistochemistry in the spinal cord during three different stages of motor neuron disease progression. A, presymptomatic stage; inset shows early microglial fusion in spinal cord. B, disease onset; C, end stage; D, wild type control. Note the dramatic increase in microglial staining with OX-42 during onset (B) and its subsequent decline during end stage (C). Scale bar: 200 μm. E, morphometric quantification of microglial immunostaining with OX-42 during disease development; * p < 0.05 and ** p < 0.001 with respect to age-matched controls; # p < 0.05 with respect to onset group.

Figure 2

OX-42 immunohistochemistry during symptomatic phase of disease. A, low power view reveals intensified immunoreactivity in spinal cord ventral horns; multiple large, rounded spots are visible. B, higher power view of large immunoreactive spots is suggestive of phagocytic clusters. C, same field as in B; counterstaining with cresyl violet facilitates identification of large immunoreactive spots as multinucleated giant cells. D, E, the same microscopic field prior to and after cresyl violet counterstaining reveals a well-formed multinucleated giant cell of the Langhans type. F, enlargement of framed area in C shows apoptotic microglial nucleus (arrow) within a giant cell. Scale bars: 500 μm (A), 40 μm (B, C), 20 μm (D-F).

Figure 3

OX-42 immunohistochemistry during end stage disease demonstrates extensive microglial cytoplasmic fragmentation (A-E). A, D, two different views of spinal ventral gray matter demonstrate loss of microglial cell integrity and widespread punctate staining indicative of cytorrhexis. Note that many neurons remain stained with cresyl violet. B, enlargement of framed area in A shows detail of microglial cytorrhexis, including a disintegrating giant cell on the right. E, enlargement of framed area in D shows detail of microglial cytorrhexis. C, motor neuron in SOD1^G93A rat reveals intense hyperchromasia with cresyl violet and nuclear cap. F, normal motor neuron and microglia from wild type spinal cord. Scale bars: 40 μm (A, D); 20 μm (B, C, E, F).

Figure 4

Lectin staining of microglia in the brainstem (level of cranial nerve VII) in wildtype animals (A) and in late symptomatic/end stage animals (B-H). Cresyl violet counterstain. A, microglia show normal ramified morphology. B, a large lectin-positive aggregate of fused microglia is evident in severely vacuolated brainstem tissue. Note enlarged perineuronal spaces to the right. C, string-like microglial fusions extend over long distances. D, breakage of neuronal process, probably a dendrite, from cell body within markedly vacuolated space (arrows). E, two multinucleated microglial giant cells are seen below a neuron with broken off process (arrow). F, large multinucleated giant cell displaying vacuolization is present amidst numerous microglial cytoplasmic fragments. G, multinucleated giant cell of the Langhans type displaying characteristic peripheral arrangement of nuclei. H, rounded lectin-positive microglial cell (arrow) within vacuolated space displays nuclear fragmentation indicative of apoptosis. Scale bars: 20 μm (A-H).

Figure 5

Visualization of microglia in midbrain with GSA-I-B₄ lectin (A-F) and in motor cortex with OX-42 (G) and OX-6 (H) during symptomatic disease. A, low power view of midbrain reveals enhanced lectin staining in the red nucleus. B, higher magnification shows that enhanced lectin reactivity is confined strictly to red nucleus region (arrows indicate perimeter of red nucleus). C, microglial fusions are interspersed with rubrospinal neurons that appear undamaged. D, lectin-positive microglial fusion (giant cell) within red nucleus. E, oculomotor nucleus reveals normal-appearing motor neurons and lack of microgliosis. F, substantia nigra (pars compacta) shows presence of normal, ramified microglial cells. G, motor cortex shows normal, ramified microglia. H, single, ramified microglial cell positive with OX-6 (arrow) near lateral ventricle. Scale bars: 400 μm (A); 200 μm (E); 100 μm (B,H); 50 μm (C,F,G); 20 μm (D).

Figure 6

Schematic depicting the approximate time course of motor neuron disease development and the accompanying microglial changes in SOD1^G93A rats. Note that disease onset and subsequent development of end stage disease is variable among individual animals.