DEPARTMENT OF OPHTHALMOLOGY
EHIME UNIVERSITY SCHOOL OF MEDICINE
TOON, EHIME 791-0295, JAPAN
Phone: (81) 89-9605361, Fax: (81) 89-9605364
Xiaodong Zheng, MD PhD
E-mail: xzheng@m.ehime-u.ac.jp
Ms April Gerobin
The BioMed Central Editorial Team
May 23, 2012
RE: MS: 1974007221687930
In Vivo Confocal Microscopic and Histological Findings of Unknown Bullous Keratopathy
Probably Associated with Pseudoexfoliation Syndrome
Reviewer's report:
This clinical note reported two cases of PEX bullous keratopathy with pathological and
confocal observation. Description is clear, and this manuscript provides us a lot of
information about PEX. The reviewer has several question as following:
(1) Main point the reviewer is interested in is confocal and pathological observation. Overall,
the authors should provide us more detailed description in case report or legend. For
example, in Figure 1F and 2D, the authors demonstrated electron microscopic photos. I
cannot identify the PEX substance in those figures. Adding arrows (or arrowhead) is
recommended. In the confocal observation in Figure 1B, 1C and 2C, PEX substance was not
directed. Detailed description about PEX substances observed by confocal microscopy is
very important. The authors’ observation would be the first description about PEX
by confocal microscopy.
RESPONSE: Agree. For clarity and easy understanding for the readers, we have modified
this manuscript and added some details in the figure legend as follows, 1) open arrows are
added in the SEM photos to show PEX fibers in close vicinity of the destroyed endothelia in
Figure 1F and 2D; 2) arrows are added in Figures 1B, 1C and 2C to show PEX materials.
(2) Confocal images requires scale bar.
RESPONSE: Agree. Each image frame is in its original size of 400μm x 400μm, scale bars
are now added in the revised manuscript for confocal images.
(3) The authors described that endothelial cells changed into fibroblastic cells. However, only
this pathological observation cannot demonstrate fibroblastic cells. Remove this description
or add figures showing fibroblast markers are expressed in this cell.
RESPONSE: Agree. This description has been now rephrased to avoid confusing in the
revised manuscript. (Page 4 line 10)
(4) The authors described thickened Descemet membrane in PEX keratopathy. If this was
not previously reported, the reviewer strongly recommends the authors to observe Descemet
membrane in PEX corneas.
RESPONSE: Descemet membrane thickening is known as one characteristic change of the
PEX keratopathy (Nauman et al in 1998). We have also noted the similar changes in PEX
eyes by light and electronic microscopy. Study in undergoing to characterize this change
with other clinical phenotypes of this disorder. Detailed description of the Descemet
membrane changes is beyond the scope of this case report.
(5) In Figure 1H, the authors showed immunofluorescence of LOXL1. But this image is not
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reliable. The LOXL1 positive lesion in the stroma seems to be positive, however, similar
lesion was observed in anterior chamber. If the authors require to present this figure, the
negative control must be required to guarantee experimental procedures.
RESPONSE: As we have shown in this figure, LOXL1 staining was noted in the stroma. The
positive staining in the anterior chamber close to endothelial layer is probably caused by
PEX materials attaching to the endothelia or by destructed endothelial cellular debris
combining PEX components. We have double-checked the slides and reconfirmed our
staining procedure. For clarity and better quality of the images, in the revised manuscript, a
new image was used in combination with a negative control as requested by the reviewer.
(6) PEX is a systemic disease thus fellow eye may be affected. The authors would
demonstrate specular microscopic images if possible.
RESPONSE: Agree. PEX fellow eyes share similar findings with their clinically affected eyes.
We have published our detailed observation of both eyes in unilateral PEX using in vivo
confocal microscopy (Zheng, IOVS 2011). Due to the space limitation of a case report as
required by the journal, we did not provide information on the fellow eyes, instead, we added
the following comments in the revised manuscript, “Alert should be raised to this unique
clinical entity that relates to aging process, bilaterally involved and is probably more
prevalent than we have believed.”(Page 6 line 6)
(7) The authors described that PEX substance was found in the corneal stroma in discussion.
If this is true, how PEX substances “migrate” to subbasal cell layers through corneal stroma?
RESPONSE: Using in vivo confocal microscopy, we and others have demonstrated the
existence of PEX materials in subepithelial layer of the cornea of PEX eyes (Martone, Clin
Exp Ophthalmol 2007; Zheng, IOVS 2011). PEX is a systemic disorder of microfibrillopathy,
subbasal layer of the corneal epithelium and Descemet membrane are all possible site of
PEX material production. Extracellular matrix components, such as basement membrane
components, may possibly interact and become incorporated into the composite PEX
material (the basement membrane hypothesis). In addition, although direct evidence is
lacking in literature, even endothelial cells are believed to be possible source of PEX
material production. Therefore, the PEX substances do not necessarily “migrate”, but a
bunch of corneal cells and basement membrane in the cornea are all possible candidates for
PEX fiber formation.
(8) Page 5 line 4, “last” should be “latest”.
RESPONSE: Agree. We have reworded this in the revised manuscript. (Page 5 line 4)
Again, we would like to express our appreciation to all of the reviewers for their
invaluable advices and important comments. Thank you.
Sincerely yours,
Xiaodong Zheng MD, PhD
Associate Professor
Department of Ophthalmology
Ehime University School of Medicine
JAPAN
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