Axonal swellings and spheroids: a new insight into the pathology of neurocysticercosis

Abstract

Neurocysticercosis is a parasitic brain disease caused by the larval form (Cysticercus cellulosae) of Taenia solium and is the leading cause of preventable epilepsy worldwide. However, the pathophysiology and relation to the wide range of clinical features remains poorly understood. Axonal swelling is emerging as an important early pathological finding in multiple neurodegenerative diseases and as a cause of brain injury, but has not been well described in neurocysticercosis. Histological analysis was performed on human, rat and porcine NCC brain specimens to identify axonal pathology. Rat infection was successfully carried out via two routes of inoculation: direct intracranial injection and oral feeding. Extensive axonal swellings, in the form of spheroids, were observed in both humans and rats and to a lesser extent in pigs with NCC. Spheroids demonstrated increased immunoreactivity to amyloid precursor protein and neurofilament indicating probable impairment of axonal transport. These novel findings demonstrate that spheroids are present in NCC which is conserved across species. Not only is this an important contribution toward understanding the pathogenesis of NCC, but it also provides a model to analyze the association of spheroids with specific clinical features and to investigate the reversibility of spheroid formation with antihelminthic treatment.

Keywords: APP; T. solium oncospheres; neurocysticercosis; neurofilament; spheroids.

Figure 1

Images showing pathologic changes surrounding T. solium cysticercosis in rat brain. In H & E images rats have well‐preserved tissue and neurons (indicated by an arrow in sham brain). Spongy changes are observed in rats infected by both intracranial and oral feeding (arrow in IC route), and show an accumulation of inflammatory cells adjacent to the collagen layer in both routes of infection. There is an intense reaction by reactive astrocytes (fibrillary gliosis), activated microglia and phagocytes in the brain tissue surrounding cysticerci of both routes of infection. Astrocytes are restricted to areas beyond the fibrotic layer while macrophage cells invade the layer of fibrosis. Inflammatory cells and gliosis were observed up to 300 μm away from the cysticercus. Scale bars: for 10× objective = 1 mm and for 40× = 50 μm.

Figure 2

Images showing rat axonal swellings (spheroids) surrounding T. solium cysticercus. Spheroids were detected using IHC to neurofilament (phosphorylated (p‐NFp), non‐phosphorylated (np‐NFp) or pan), ubiquitin, SOD1 or Hsp70 antibodies. No spheroids were found in sham animals. The images show spheroids located in cortical areas adjacent to the cysticercus for the intracranial (IC) and oral routes of infection but were also found in the hippocampus (data not shown). Spheroids were more clearly observed using NFp than with np‐NFp or pan‐NFp antibody. Ubiquitin and SOD1 in sham sections were restricted to the cytoplasm as expected. In both IC and oral routes of infection, ubiquitin and SOD1 spheroids were scattered in the cortex and associated with spongy changes. Hsp70 spheroids were fewer compared to other markers. Scale bars = 50 μm.

Figure 3

Spheroid distribution in intracranially infected rats. The graph shows a kernel density plot of the different spheroid markers. Distance represents the extension of the different markers from the fibrotic layer of the cysticerci. Most spheroids are found in conjunction with spongy changes (600 um) and gliosis but extend more than 1500 um into the tissue that appears to be otherwise normal.

Figure 4

Spheroids shown by confocal imaging. Sham sections showed the normal distribution of SOD1, neurofilament forms, ubiquitin and Hsp70, with no spheroids seen. Infected brains (intracranial route) show most neurofilament protein (p‐NFP200, np‐NFP200KDa and pan‐NFP200KDa) forms do not overlap with SOD1. However, as is shown in SOD1 and p‐NFP200KDa immunofluorescence, SOD1 spheroids can be surrounded by or can coexist with NFp. SOD1 and ubiquitin double labeling shows that spheroids share a similar distribution as well as SOD1 and Hsp70 spheroids.

Figure 5

Spheroids found in human, rat and pig NCC. Amyloid precursor protein (APP) staining shows that spheroids are present in human and rat NCC. Cases 1–3 demonstrate axonal swellings while in case 4 axonal swellings are less conspicuous among inflammatory changes. Pig samples do not show evidence of spheroids positive for APP (not shown) but axonal swellings were detected with high/medium‐MW neurofilament (HM NfP) IHC.