Original Investigation
Punctal Stenosis: Histopathology, Immunology, and
Electron Microscopic Features—A Step Toward Unraveling
the Mysterious Etiopathogenesis
Mohammad Javed Ali, F.R.C.S.*, Dilip Kumar Mishra, M.D.†, Farhana Baig, M.D.‡,
Mekala Lakshman, Ph.D.§, and Milind N. Naik, M.D.*
*Dacryology Service and †Ocular Pathology Service, L.V. Prasad Eye Institute; ‡Department of Pathology, Global
Hospitals; and §Ruska Laboratories, College of Veterinary Science, Hyderabad, India
Purpose: To study the histologic, immunohistochemical, and
electron microscopic features of puncta and proximal vertical
canaliculi to understand the etiopathogenesis of punctal stenosis.
Methods: Prospective study of 26 stenosed punctae that
were collected following a punctoplasty. Sixteen were from
lower eyelid and 10 from upper eyelid. Histopathological
examination was performed on 20 punctae using hematoxylin-
eosin, periodic acid-Schiff, and Masson trichrome staining.
Immunohistochemical patterns were analyzed after staining
with leukocyte common antigen or CD45, CD3, CD5, CD10,
CD20, CD138, and smooth muscle actin. Six punctae (3 upper,
3 lower) were separately processed for electron microscopic
studies as per standard protocols.
Results: All punctae showed evidence of subepithelial and
subconjunctival fibrosis. Thirty percent (6/20) showed extensive
fibrosis. Inflammation was noted in 80% (16/20) of the samples;
however, 20% (4/20) showed severe inflammation. Strong
immunoreactivity was noted, with CD45 and CD3 in 80%
(16/20) with predominance in the subepithelial areas. Focal
immunoreactivity was noted for CD10, CD20, and CD138.
Immunoreactivity was negative for CD5. Electron microscopic
features include blunted epithelial microvilli, numerous
fibroblasts, extensive and irregularly arranged collagen bundles,
mononuclear infiltration in the vicinity of fibroblasts or in
between collagen bundles, and inter- and intracellular edema in
areas of inflammation.
Conclusions: Chronic inflammation and subsequent fibrosis
appear to be the basic ultrastructural response to various noxious
stimuli. Mononuclear inflammatory infiltration in the vicinity
of fibroblasts could possibly reflect a close cellular interaction
between these 2 cells.
(Ophthal Plast Reconstr Surg 2015;31:98–102)
Accepted for publication April 14, 2014.
The authors have no financial or conflicts of interest to disclose.
This study has been reviewed by the ethics committee and has been
performed in accordance with the ethical standards laid down in the 1964
Declaration of Helsinki. Informed consent was obtained from the patients.
M.J.A. helped in concepts and manuscript writing; D.K.M. in pathological
examination and correlation; F.B. in electron microscopic work and
manuscript review; M.L. in electron microscopic work; and M.N.N. in
critical inputs and review.
Address correspondence and reprint requests to Mohammad Javed Ali,
F.R.C.S., L.V. Prasad Eye Institute, Road No 2, Banjara Hills, Hyderabad 34,
India. E-mail: drjaved007@gmail.com
DOI: 10.1097/IOP.0000000000000204
98 Ophthal Plast Reconstr Surg, Vol. 31, No. 2, 2015
P unctal stenosis is a common disorder of the punctum. It is
an important cause of epiphora and accounted for 8% of
all patients presenting with epiphora in a tertiary care oculo-
plastics practice.1 Numerous causes are known, which can be
broadly grouped under inflammatory, infective, traumatic, and
drug toxicity.2–8 Although the pathogenesis is still elusive, it is
important to remember that punctum being the entry point for
tears is exposed to all the possible soluble irritants that an ocu-
lar surface encounters. The widely believed hypothesis that has
been supported by a single good histologic study9 is a common
mechanism involving inflammation leading to fibrosis and sub-
sequent stenosis. Deficiencies and lacunae in current knowledge
regarding punctal stenosis include insufficient histopathological
information, lack of immunophenotyping of inflammatory cells,
unknown ultrastructural changes, and unknown cytokine media-
tors and their receptor expressions. The present study describe
histopathological features, immunohistochemical typing of
inflammatory cells, and electron microscopic features of punctal
stenosis as a step further in efforts to unravel the pathogenesis.
MATERIALS AND METHODS
The prospective study involves 26 stenosed punctae collected
following a punctoplasty for epiphora. Sixteen were from lower eyelid
and 10 from upper eyelid. No patients had associated canaliculitis. All
patients were operated by a single surgeon (M.J.A.). Institutional re-
view board and ethics committee approval was obtained. All patients
provided an informed written consent. Histopathological examination
was performed on 20 punctae using hematoxylin-eosin, periodic acid-
Schiff, and Masson trichrome staining. Masson trichrome was specifi-
cally used to study the collagen patterns and fibrosis, which are stained
blue. Immunohistochemical patterns were analyzed after staining with
leukocyte common antigen (LCA) or CD45, CD3, CD5, CD10, CD20,
CD138, and smooth muscle actin (SMA). Six punctae (3 upper, 3 lower)
were separately processed for electron microscopic studies as per stan-
dard protocols.
Transmission Electron Microscopy (TEM) Protocol. The samples
were fixed in 2.5% glutaraldehyde in 0.1-M phosphate buffer (pH 7.2)
for 24 hours at 4°C and postfixed in 2% aqueous osmium tetroxide in
0.1-M phosphate buffer for 2 hours. The tissues were dehydrated in a
series of graded alcohols, and infiltrated and embedded in Araldite 6005
resin or Spur resin. Ultrathin sections of 50 nm were made with a glass
knife using the Ultra-microtome (Lieca Ultracut UCT-GA-D/E-1/00,
Wetzlar, Germany). The ultrathin sections were then mounted on cop-
per grids and stained with saturated aqueous uranyl acetate and coun-
terstained with Reynold’s lead citrate. The sections were viewed under
TEM (Hitachi H-7500, Tokyo, Japan) at appropriate magnifications as
per standard protocols.10
Ophthal Plast Reconstr Surg, Vol. 31, No. 2, 2015 Pathogenesis of Punctal Stenosis

RESULTS Electron Microscopic Features. The epithelial cells appeared normal

The universal histopathological and ultrastructural feature noted
in all the punctae was the presence of fibrosis involving the subepithelial
areas of both canalicular and conjunctival epithelium (Fig. 1A–F). The fi-
brosis was extensive in 30% (6/20) of the samples. This finding was amply
evident by the Masson trichrome staining (Fig. 1C,D, and F). Inflammation
was noted in 80% (16/20) of the samples; however, 20% (4/20) showed
severe inflammation (Fig. 2A–D). Only 15% (3/20) showed focal areas of
squamous metaplasia. Immunoreactivity was positive for SMA (Fig. 3A),
showing numerous fibroblasts. There was a strong immunoreactivity with
LCA or CD45 and CD3 in 80% (16/20), with predominance in the sub-
epithelial areas (Fig. 3B,C). This reflects the presence of plenty of T cells.
Focal immunoreactivity was seen with markers CD10, CD20, and CD138
(Fig. 3D,E), which reflects the focal presence of B cells, plasma cells, and
occasional lymphoid progenitors ready to differentiate into T cell, B cell,
or natural killer (NK) cell. All the samples were negative for CD5, which
indicates lack of acute inflammation (Fig. 3F).
with tight intercellular junctions with an occasional goblet cell in be-
tween (Fig. 4A). Mitochondria were numerous. The nuclei were elon-
gated with patchy or peripheral chromatin condensation near the inner
nuclear membrane. There were numerous slightly blunted microvilli
toward the luminal side (Fig. 4A). Areas without dense fibrosis showed
fibroblasts with increased but regular collagen bundles (Fig. 4B). Pleo-
morphic mitochondria with occasional peripheral matrix condensation
and dilated endoplasmic reticulum were noted. Few fibroblasts also
showed nuclear haloes (Fig. 4B). In punctae with dense fibrosis, nu-
merous fibroblasts were noted along with extensive and irregularly ar-
ranged collagen bundles (Fig. 4C,D). This irregular arrangement was
widespread and noted in both the longitudinal and the cross-sectional
bundles (Fig. 4C). Mononuclear inflammatory infiltration was noted
distinctly in 2 areas: one among the collagen bundles (Fig. 4E) and the
other in the vicinity of fibroblasts (Fig. 4F). Both areas showed features
of edematous spaces (Fig. 4E,F).

FIG. 1.  Fibrosis in punctal stenosis. Microphotograph showing dense fibrosis with fibroblasts beneath the canalicular epithelium
(hematoxylin-eosin, ×400) (A). Widespread fibrosis beneath the focally metaplastic canalicular epithelium (Masson trichrome, ×100)
(B). High magnification of subepithelial canalicular tissues showing areas of less dense fibrosis (Masson trichrome, ×400) (C). Sub-
epithelial canalicular tissues showing areas of dense fibrosis (Masson trichrome, ×400) (D). Areas of dense fibrosis with inflammation
beneath the conjunctival epithelium (hematoxylin-eosin, ×100) (E). Widespread subconjunctival fibrosis with sparse cellular infiltration
(Masson trichrome, ×100) (F).

© 2014 The American Society of Ophthalmic Plastic and Reconstructive Surgery, Inc. 99
M. J. Ali et al. Ophthal Plast Reconstr Surg, Vol. 31, No. 2, 2015

FIG. 2.  Chronic inflammation in punctal stenosis. Microphotograph showing inflammatory infiltrate in vicinity of canalicular epithe-
lium (hematoxylin-eosin, ×400) (A). Inflammatory infiltrate along with fibrosis below the canalicular epithelium (Masson trichrome,
×400) (B). Marked inflammatory infiltrate below the conjunctival epithelium (hematoxylin-eosin, ×400) (C). Inflammatory changes in
subconjunctival tissues with less dense fibrosis (Masson trichrome, ×100) (D).

DISCUSSION differentiate to T cell, B cell, or an NK cell. CD5 is expressed in

Punctal stenosis is a common cause of epiphora. Patients
1 a specific IgM secreting subset of B cell or a strongly stimulated
undergoing punctoplasty have predominantly been older women
with a mean age of 65 years.2–9 Associations of punctal steno-
sis include blepharitis, chronic conjunctivitis, meibomian gland
dysfunction, and eyelid malpositions.2–9 The preponderance of
inflammatory etiologies, associated inflammatory pathologies
in the vicinity, and histopathological evidence points toward a
common mechanism of chronic inflammation and fibrosis that
progressively leads to narrowing of the punctum.
Port et al.9 studied histopathological features of punctal
stenosis in 30 eyes of 24 patients. Most of their patients (87.5%)
were operated for epiphora and the remaining for managing
Actinomyces canaliculitis. Chronic inflammatory features were
noted in 36.7%, fibrosis in 23.3%, chronic inflammation and
fibrosis together in 13.3%, normal conjunctival epithelium in
10%, and squamous metaplasia in 10% of the eyes. The cur-
rent study found fibrosis to be a universal feature, with 30%
having extensive fibrosis. Inflammation was noted in 80% and
squamous metaplasia in 15% of the samples. The current study
went a step further to type the immune cells by immunohis-
tochemistry and documents the cellular and subcellular effects
using TEM.
Immunohistochemical studies were carried out using the
CD markers. CD stands for cluster of differentiation and is a
protocol used in immunophenotyping of the cells by identifying
certain cell surface molecules. CD45 or LCA is a nonspecific
marker expressed on the cell surface of all the leukocytes. T lym-
phocytes are CD45+ and CD3+, which were strongly positive in
80% of samples in the current study. B lymphocytes are CD45+
and CD20+, which were focally immunoreactive in the punc-
tal stenosis samples. Plasma cells can be identified by CD138.
CD10 is expressed in common lymphoid progenitors, which can
active T cell. Both CD138 and CD10 were expressed focally in
subepithelial areas, whereas no CD5 expression could be noted
in the current study. All this reflects predominant cellular infil-
tration by T cells and chronic inflammation.
Electron microscopic features of punctal stenosis mainly
included blunted epithelial microvilli, numerous fibroblasts,
extensive and irregularly arranged collagen bundles, mono-
nuclear infiltration in the vicinity of fibroblasts or in between
collagen bundles, and inter- and intracellular edema in areas of
inflammation.
In conclusion, the current study and its findings enhance
the understanding of the pathophysiological mechanisms
involved in punctal stenosis. Further exploration by molecular
biology techniques to zero in on the various cytokine receptor
expression in punctal tissues and their cellular mediators in the
tears would help unravel the etiopathogenesis of this enigmatic
disorder.
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100 © 2014 The American Society of Ophthalmic Plastic and Reconstructive Surgery, Inc.
Ophthal Plast Reconstr Surg, Vol. 31, No. 2, 2015 Pathogenesis of Punctal Stenosis

FIG. 3.  Immunohistochemical typing in punctal stenosis. Microphotograph showing immunohistochemical patterns of positive staining
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M. J. Ali et al. Ophthal Plast Reconstr Surg, Vol. 31, No. 2, 2015

FIG. 4.  Electron microscopic features. Transmission electron micrograph (TEMG) showing epithelial cells (E), their nuclei (N), goblet
cell (G), and microvilli (M) (×6,755) (A). TEMG showing less dense fibrotic areas with the fibroblast (F) surrounded with increased but
neat collagen bundles. There is evidence of nuclear halo (NH), pleomorphic mitochondria (M), and dilated endoplasmic reticulum (ER)
(×15,440) (B). TEMG of dense fibrotic areas showed both the longitudinal (Co) and cross-sectional bundles to be extensive and irregu-
lar with edematous areas (E) and one artifact (A). (×7,720) (C). Higher magnification TEMG showing a compressed fibroblast (F) with
dense and irregular collagen bundles (Co) (×9,650) (D). TEMG showing mononuclear inflammatory infiltrate (L) within the collagen
bundles (Co) with intervening edematous spaces (E) (×3,860) (E). TEMG showing mononuclear infiltration (L) in vicinity of fibroblasts
(F) (×11,580) (F).

102 © 2014 The American Society of Ophthalmic Plastic and Reconstructive Surgery, Inc.