Journal Club: Macrophages on and below the basilar membrane: activation and migration during ototoxic damage response.

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Today's journal article

Ye M, Zhang C, Ding D, Chen GD, Adler HJ, Sharaf R, Hu BH. Organ of Corti macrophages: a distinct group of cochlear macrophages with potential roles in supporting cell degeneration and survival. 

Why I picked this article

Our hearing organ, the cochlea, is thought to have its own immune system. An important player in the immune system is the "macrophage".  Macrophages remove debris and fight infection. In the cochlea, macrophages are normally present in various areas, such as the spiral ligament. When injury occurs, more immune cells arrive from the bloodstream to clean up damage and support repair, as well as cochlear macrophages becoming more "activated". 

The organ of Corti is a special region within the cochlea that contains sensory and supporting cells crucial for hearing. Cells within the organ of Corti, as well as the environment surrounding it, are very delicate, and it’s easily damaged by noise, drugs, genetic factors, or ageing, leading to permanent hearing loss. There are some questions that still remain around how/if immune response directly impacts the organ of Corti, and if immune cells like macrophages respond to injury in this region. 

This study investigated how macrophages respond to drug-induced and age-related cochlear damage, focusing on those within the organ of Corti, using high-dose cyclodextrin—a compound safe at low levels but ototoxic at high doses—as a controlled model for inner ear injury.

Some of the research findings

Mouse model: 

  • Healthy group: CBA/CaJ mice (1.5–4 months old)
  • Aged group: C57BL/6J mice (17–19 months old)
  • Acute ototoxicity group: cyclodextrin ototoxicity in CBA/CaJ mice, 1–4 days after treatment
  • Chronic ototoxicity group: cyclodextrin ototoxicity 2–10 weeks after treatment
  • cyclodextrin = 2-Hydroxypropyl-b-cyclodextrin, 8 mg per g body weight. 
Markers for immune cells for immunochemical detection. 

  • CD45 Antibody = Pan-leukocytes (AF114, R&D System)
  • Iba1 Antibody = Macrophages (ab178846, Abcam Inc.)
  • CD68 Antibody = Macrophages (MCA1957GA, Bio-Rad)
  • Galectin-3 Antibody = Macrophages and Hensen’s cells (AF1197, Novus Biologicals)
  • IFIT3 Antibody = Deiters’ cells and pillar cells (PA522230, Invitrogen)
Findings: 
  • Of the two sensory cells, outer hair cells and inner hair cells, cyclodextrin treatment induced outer hair cell loss in the high-frequency region and mid-frequency zones, while inner hair cells were lost primarily in the high-frequency regions of the cochlea during the acute phase. 
  • Cyclodextrin treatment caused further sensory cell death, particularly inner-hair cell loss, in the chronic phase. 
  • Macrophages (defined here as Iba1- and CD45-positiveimmunecells) were identified on the organ of Corti. 
    • Organ of Corti macrophages were located above the basilar membrane
    • Basilar membrane macrophages were located below the basilar membrane (on scala tympani side)
  • The number of basilar membrane macrophages increased during acute phase of ototoxic injury. 
  • Organ of Corti macrophages increased especially from the high-frequency zone in the chronic phase of ototoxic injury. 
  • CD68 protein increased in the organ of Corti macrophage after ototoxicity, compared to the control. 
  • Both the organ of Corti macrophage and basilar membrane macrophages were found, and had high CD68 protein levels in the ageing animal cochlea. 
"Notably, the presence of OC macrophages was spatially correlated with supporting cell pathogenesis."

Part of Figure 6B: The Basilar membrane macrophages (arrow) after chronic ototoxicity.

Haruna's takeaway

Very nice immunohistochemistry visualisation of macrophages, using whole mount and 3D imaging. It is interesting to see the variation in the shape and location of macrophages. Subclassification of macrophages into the organ of Corti macrophages and basilar membrane macrophages, primarily by their location, is a good idea and makes it simple, in terms of counting their numbers or comparing morphologies. I will keep this in mind when we look at the macrophages. 

Microscopy-based understanding of macrophages to get snapshots of where macrophages are and how they appear is great, but it also highlights the challenge of not being able to monitor some of these macrophage movements live. Don't we wish we could simply see macrophages move in time, to understand where they come from and how fast they start reacting? Only if! One thing to note is that there are capillaries on the basilar membrane. It may be interesting to investigate macrophage distribution relative to these small blood vessels, to see if these vessels could be some kind of highway or macrophage migration road. 

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This is Haruna's 30/100 of the 100-day challenge to post a science blog article every day! I love inner ear biology & cochlear physiology.