Download Mast cells in human optic nerve.

Mast Cells in Human Optic Nerve
Leonard A. Levin,*§ Daniel M. Albert,^ and David Johnson\%
Purpose. Mast cells are classically found in ocular tissues within the conjunctiva, choroid, and
iris. The aim of this study was to examine their distribution in the optic nerve and its meninges.
Methods. Sixty-six human optic nerves were studied from normal subjects at autopsy, fetuses
aborted for chromosomal abnormalities, and from enucleation specimens of patients with a
variety of inflammatory, traumatic, neoplastic, and vascular disorders. Mast cells were identified using a stain for the enzyme chloracetate esterase, and confirmed using toluidine blue,
revealing metachromatic cytoplasmic granules.
Results. Mast cells were found scattered in the meninges of almost all optic nerves examined,
frequently in perivascular locations, with densities up to 2325 mast cells/mm3 (mean 269.7 ±
64.1 cells/mm3 in normal nerves). Mast cells were found in the optic nerve parenchyma in
nerves from four eyes that had severe abnormality, often associated with neovascularization.
Normal nerves, as well as nerves from fetuses aborted for congenital defects, had significantly
fewer meningeal mast cells than those from eyes with inflammatory or vascular diseases. Degranulation of mast cells was observed more often in eyes with recent severe trauma.
Conclusions. Based on their work and the work of others suggesting an association between
mast cells and nervous system autoimmune disorders, the authors hypothesize a role for optic
nerve mast cells in certain ocular inflammatory conditions. Invest Ophthalmol Vis Sci.
1993;34:3147-3153.
JVlast cells are widely distributed, especially in connective tissue and mucosal surfaces. Although their
role in normal physiological functioning is not known,
they play a central role in allergic responses through
the release of a number of inflammatory mediators.
Mast cells can be stimulated both in an antigen-specific
manner through binding to cell-surface immunoglobulin E, and nonimmunologically.1 In the eye, they are
classically found in the conjunctiva, choroid, ciliary
body, and iris; it has been theorized that they are involved in the pathophysiology of autoimmune uveitis.2
Workers in our laboratory have been studying the role
of mast cells in experimental autoimmune demyelinating disease and have found that susceptibility to experimental autoimmune encephalomyelitis and experimental autoimmune neuritis correlate with the number of dural and sciatic nerve mast cells, respectively.3
Experimental autoimmune neuritis develops less
readily in animals treated with the mast cell stabilizer
nedocromil,4 and mast cells release proteases that can
degrade myelin.5 As part of an understanding of the
pathophysiology of events that may underlie inflammatory processes in the optic nerve, such as optic neuritis, we examined optic nerves for the presence of mast
cells, using a stain for the enzyme chloracetate esterase, and with toluidine blue. We present here the first
systematic study of the pattern and distribution of
mast cells in human optic nerves from normal subjects, as well as subjects with a variety of ocular disorders.
METHODS
From the *Department of Ophthalmology, Massachusetts Eye and Ear Infirmary, the
•f Department of Ophthalmology, University of Wisconsin at Madison, the %Cenler
for Neurologic Diseases, lirigham and Women's Hospital, and § Harvard Medical
School, Boston, Massachusetts.
Submitted for publication October 8, 1992; accepted March 19, 1993.
Proprietary interest category: N.
Reprint requests: Leonard A. Levin, MD, PhD, Department of Oplhalmology,
University of Wisconsin Hospital, F4¥=340 CSC, 600 Highland Avenue, Madison,
Wl 53792.
Sections from sixty-six human whole eyes previously
submitted for pathological diagnosis after surgery or
autopsy from subjects with either documented eye disease or no history of ocular pathology were studied
(Table 1). The tenets of the Declaration of Helsinki
were followed. All tissue used was obtained subsequent to pathological examination, and all patients
undergoing enucleation had previously given in-
Investigativc Ophthalmology & Visual Science, October 1993, Vol. 34, No. 11
Copyright © Association for Research in Vision and Ophthalmology
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Investigative Ophthalmology & Visual Science, October 1993, Vol. 34, No. 11
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TABLE
l. Optic Nerve Mast Cell Density
Number
Group
Normal
Anomalies*
Neoplasmf
Inflammatory]:
Phthisis§
Trauma"
VascularU
of Eyes
20
7
12
10
4
6
7
Mean Nerve
Length (mm)
Mast Cells
(per mm3)
269.7 ±64.1
15.2 ±5.6
341.8 ± 74.2
676.2 ±210.6
392.1 ± 162.5
482.3 ± 98.6
595.8 ± 140.1
6.7
3.6
5.6
5.3
7.2
5.6
4.9
Degranulation
(%)
4.7 ± 1.9
0.0 ±0.0
13.6
4.8
18.9
44.7
6.0
±5.2
±2.4
± 14.3
± 14.8
±3.9
Counts of mast cells within optic nerves from patients with various disorders, using the chloroacetate esterase staining method.
* Multiple congenital anomalies, Klippel-Trenaunay syndrome, multiple skeletal anomalies, trisomy 13, trisomy 18.
f Choroidal melanoma, retinoblastoma, carcinomatous meningitis of nerve, retinal pigment epithelium tumor.
X Cytomegalovirus, granulomatous uveitis, panuveitis, herpes zoster ophthalniicus, oculocutaneous pemphigoid and endophthalmitis, necrotizing scleritis, toxocara.
§ Endstage diabetic retinopathy, chronic retinal detachment, retinopathy of prematurity.
11
Intraocular foreign body, ruptured globe.
U Branch retinal artery occlusion, branch retinal vein occlusion, central retinal vein occlusion, ischemic oculopathy.
formed consent to the use of tissue that was not
needed for pathological diagnosis for research purposes. The eyes were fixed in formalin, embedded in
paraffin, and sectioned according to routine methods.
All had at least 1 mm of optic nerve present. Individual
sagittal sections containing the optic disk and contiguous nerve were incubated with naphthol AS-D chloroacetate, a substrate specific for the chloroacetate
esterase in mast cells and neutrophils, according to
modifications of an established protocol.6 Briefly, a
0.4% solution of naphthol AS-D chloroacetate (Sigma
Chemical, St. Louis, MO) in 1 ml N,N-dimethyl formamide was diluted in 40 ml of 0.067M phosphate
buffer (pH 7.4). To this was added a 1:1 mixture of 0.1
ml of 4% sodium nitrite in distilled water and 0.1 ml of
4% New Fuchsin (Allied Chemical, Morristown, NJ) in
2 N hydrochloric acid. After careful mixing for 60
seconds, the solution was filtered and used to incubate
hydrated paraffin sections of optic nerve for 45 minutes at room temperature. After washing and counterstaining with hematoxylin, sections were dehydrated
and mounted. Mast cells were identified by their single
rounded nucleus, the presence of numerous small
brick-red cytoplasmic granules, and their characteristic oval or polygonal cytoplasmic structure. Sections of
human tonsil were used as a positive control.
To further confirm the specificity of the stain for
mast cells, separate sections were stained with acidified toluidine blue (0.15% solution in 0.25 M HC1, pH
1.2) for 15 minutes at room temperature. No counterstain was used. Mast cells were identified by their characteristic metachromatic purple granules surrounding
a light-blue nucleus, within an oval or polygonal cytoplasm.
All sections were counted by a single observer
(LL) masked to the clinical diagnosis of each subject.
Each mast cell was counted, and the distance of each
cell from the most anterior aspect of the edge of the
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optic nerve head was measured and recorded in a
computer database, as well as the length of the nerve
in the section. For the purposes of this study, degranulation was defined as the presence of extracellular
granules not enclosed by a cell membrane adjacent to
an otherwise typical appearing mast cell, or as a collection of extracellular granules focally surrounding a
ghostlike cell. In three specimens with inflammatory
cells, the areas with large numbers of these cells were
repeatedly examined to minimize confusion between
mast cells and neutrophils.
Biometric techniques were employed to estimate
the number of mast cells per unit volume. Briefly, the
number of mast cells seen in a unit area of a slide was
multiplied by the number of section thicknesses per
unit depth, and then corrected for multiple countings
by a factor dependent on the thickness of the sections
and the average size of the mast cells (Figure 1).
Student's t test was used for comparisons between
two groups and one-way analysis of variance for testing equality of multiple group means. A Poisson distribution was used to model an idealized randomly distributed mast cell population, and its fit measured with
the Kolmogorov-Smirnov statistic.
RESULTS
Of the 66 eyes stained for chloroacetate esterase, 65
had one or more mast cells present in the optic nerve
meninges or parenchyma. These were identified by the
brilliant carmine staining of their cytoplasmic granules, the characteristic shape, and densely-packed
evenly sized granules typical of connective tissue mast
cells (Figure 2). In normal nerves from eyes studied at
autopsy, mast cells were only found in the meninges.
For the most part, mast cells were distributed in the
dura of the nerves, and only rarely in the arachnoid.
There was some indication of increased concentration
Mast Cells in Human Optic Nerve
3149
RIOMFTRY
Volume of meninges = volume of outer cylinder - volume of inner cylinder
= L x n x (D/2)2 - L x n x (d/2)2
Correction factor (accounts for the fact that a single cell
may be seen on more than one thin section)
O
o—
Cells / volume = cells / area / (thickness x
FIGURE 1. Protocol for computing number of mast cells per
unit volume from counts of slide sections. In this method,
mast cells were modeled as spheres as assumed to be approximately 10 /im in diameter. The correction factor is needed to
account for the fact that a single mast cell may occur on
more than one slide, and thus be otherwise overcounted.
of mast cells adjacent to meningeal vessels (Figure 3).
When biometric techniques were used to estimate the
total number of mast cells per unit volume of tissue,
the density was as much as 2325 mast cells per mm3
with a mean of 269.7 ± 64.1 in normal nerves. There
was a wide dispersion of mast cell counts between individual specimens. Although there was no specific
correlation of mast cell number with specific disease
etiology, there was a pattern seen, with normal nerves
and nerves from fetuses aborted for congenital defects
having fewer mast cells than those with inflammatory
or vascular diseases (P = 0.026 and 0.024, respectively; Table 1). Eyes with trauma also had high numbers of mast cells, but this did not reach statistical significance. When the presence of inflammation on routine pathological examination was correlated with
disease group, all eyes with recent trauma had some
uveal inflammatory cells, which were predominantly
nongranulomatous and in the choroid. Three eyes
from patients with globe trauma, herpes zoster ophthalmicus, and endophthalmitis had low levels of optic
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nerve inflammation; in these cases, mast cells were not
particularly co-localized with areas of inflammation.
There was no association between mast cell number
and the presence of optic atrophy or gliosis. Except
for one eye with meningeal involvement from carcinomatous meningitis, there was no involvement of the
optic nerve by any of the neoplasms studied. An eye
from a patient with neovascular complications (rubeosis and neovascular glaucoma) of diabetic retinopathy
also had a high concentration of mast cells (746.7/
mm3); neither this, nor any other eye, had neovascularization of the disk visible on histologic sections.
Mast cells were only rarely seen in the parenchyma
of optic nerves (4 of the 66 patients [6.1%], Figure 4).
These patients had evidence of prior eye disorders,
specifically central retinal vein occlusion, branch retinal vein occlusion, neovascular glaucoma after
trauma, and end-stage glaucoma after retinal detachment surgery. These parenchymal mast cells were often noted adjacent to vessels, particularly the central
retinal artery and vein (Figure 5). Parenchymal mast
cells were not seen in any optic nerves from fetuses,
acutely traumatized globes, or donor eyes.
To estimate whether mast cells were randomly distributed, a histogram of distances between adjacent
mast cells was constructed, measured in a longitudinal
direction (Figure 6). The mean spacing between mast
cells in the longitudinal direction was 289 /im, with a
standard deviation of 334 /xm. To assess whether a
pattern of grouping was present, the data were compared with what would be expected if the mast cells
were distributed randomly in the anteroposterior direction. Comparison between the observed distribution and a theoretical Poisson distribution was not significant (Kolmogorov-Smirnov statistic = 0.287).
Degranulated mast cells were frequently present,
as identified with either stain by the presence of multiple extracellular granules (Figure 7). Although a
chronically degranulated cell could not theoretically
be detected, because of a lack of staining, the specimens with acute or partial mast cell degranulation
were studied. There was a correlation between type of
disease and percentage of degranulated mast cells,
with eyes with a history of recent severe trauma having
44.7% ± 14.8% degranulated cells. Eyes with intraocular neoplasms and phthisical eyes had fewer degranulated mast cells (13.6% ± 5.2% and 18.9% ± 14.3%),
whereas eyes with inflammatory and vascular disorders had about the same low level of degranulation
as was seen in normal eyes or those from fetuses with
congenital anomalies (4.7%). Analysis of variance
showed the differences between groups to be highly
significant (P < 0.001).
Studies with acidified toluidine blue reproduced
the staining pattern observed with chloroacetate esterase staining. Mast cells were readily identified by their
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FIGURE 2. Mast cells in the meninges of human optic nerve
from a patient dying of congestive heart failure. The polygonal cytoplasm, small rounded dark nucleus, and brilliant
carmine granules are characteristic of mast cells using the
chloroaceiate esterase stain. (Original magnification X 315.)
metachromatic purple granules and slightly blue
round nuclei (Figure 8). As with the chloroacetate esterase stain, mast cells were seen in the meninges of
the optic nerve, often adjacent to blood vessels.
DISCUSSION
It has long been recognized that mast cells are distributed in certain ocular tissues. In the conjunctiva, they
are partly responsible for atopic responses, such as
allergic conjunctivitis or atopic keratoconjunctivitis.7
These conditions are typically treated with agents that
FIGURE 3.
Masi cell adjacent to small vessel of the optic nerve
meninges from a 66-year-old woman with chorioretinitis of
unclear cause. The absence of a vessel near a mast cell may
be a result of the section being parallel to and displaced
from the axis of the vessel; this sectioning artifact may underestimate the number of perivascular mast cells (chloroacetate esterase; original magnification X 315).
block the physiological effect of mast cell granule contents, such as antihistamines, or with agents that block
mast cell degranulation, such as cromolyn. Mast cells
are also found in the conjunctiva, episclera, iris, ciliary
body, and choroid, where there is evidence suggestive
of a role in the pathogenesis of certain experimental
V
4. Parencyhmal mast cells in an optic nerve from a
32-year-old man with neovascular glaucoma 15 years after
trauma. There are no nearby inflammatory cells to indicate
that the cell has entered the tissue from the intravascular
space (chloroacetate esterase; original magnification X 50).
FIGURE
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FIGURE 5. Mast cells in the parenchyma of an optic nerve
from a 79-year-old woman with neovascular glaucoma after
central retinal vein occlusion. The mast cells are easily seen
adjacent to the central retinal vein {large arrow). Neutrophils
{small arrow), which also stain for chloroacetate esterase, can
be differentiated from mast cells by their distinctive cytoplasmic and nuclear morphology. In general, parenchymal
mast cells are seen far less frequently than those in the meningeal coats of the optic nerve (chloroacetate esterase; original magnification X 200).
Mast Cells in Human Optic Nerve
Grouping of Mast Cells in the
Meninges of Human Optic Nerve
k
FIGURE 6. Histogram of the distances between adjacent mast
cells measured in the longitudinal direction. The data are
not significantly different from a Poisson distribution, suggesting that there is no particular pattern of distribution of
mast cells within the anteroposterior axis of the human optic
uveitides.89 In particular, it has been shown that the
susceptibility of strains of rats to experimental autoimmune uveitis correlates with the number of mast cells
in the choroid.2 Functional studies of mast cells in
uveitis are still preliminary, however, and their role in
this condition remains to be clarified.
The results of the current study suggest that the
optic nerve meninges are a compartment for mast cells
in ocular tissue. Although there have been previous
reports of histamine of mast cell origin isolated from
bovine and rabbit optic nerves,10 to our knowledge
this is the first study that systematically studied mast
cell distribution in normal and abnormal human optic
nerves. The identification of these cells is supported by
the use of two histologic techniques, each of which is
well established biochemically. Naphthol AS-D chloroacetate is a substrate for the chloroacetate esterase
found in mast cells and neutrophils.6 Acidified toluidine blue stains the heparin and sulfated heparan
conjugates found in the granules of mast cells, causing
metachromasia. Although the chloroacetate esterase
method may stain neutrophils in addition to mast cells,
distinct morphologic differences between the two cell
types, such as the shape of the nucleus and cytoplasm,
the size and color of the granules, as well as the absence of inflammatory cells in almost all of the specimens studied make this consideration unlikely.
Mast cells in the optic nerve meninges are sparsely
distributed. In this study, their mean concentration in
normal human eyes of 269.7 ± 64.1 per mm3 compares with choroidal numbers in the rabbit of approximately 500 per mm3 (calculated from Figure 1 and
Table 2 of Levene11). In rat brain meningeal mast cell
studies, counts ranging from 10 to 60 cells per mm2 of
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3151
tissue section have been reported,3 which on a volume
basis would be approximately 1000 to 6000 cells per
mm3. Limitations of patient data collection precluded
correlating mast cell number and certain phenotypic
characteristics, particularly human leukocyte antigen
groupings. Nevertheless, genetic variation of mast cell
number may account for some of the patient-to-patient variability in mast cell number observed in these
studies. Most of the variation is accounted for by the
differences in the nature of the eye disorder. Presumptively normal nerves, such as those from fetuses
or autopsy eyes, had the lowest number of mast cells
(15.2 ± 5.6 and 269.7 ± 64.1 cells/mm3). In comparison, eyes from patients with inflammatory disorders,
such as herpes zoster ophthalmicus, panuveitis, and
Guillain-Barre syndrome, had the highest number of
mast cells. Presumably cytokines, such as interleukin3 12 and other interleukins released by inflammatory
cells involved in the disease process in these eyes
caused either migration or proliferation of mast cells,
some of which ended up in the optic nerve meninges.
Mast cell degranulation results in the release of a
variety of inflammatory mediators that may affect immunity and vascular permeability. In particular, cytokines released by mast cell degranulation, such as interleukins-3,4,5,6 and tumor necrosis factor-alpha
may potentiate local inflammation, and histamine and
other vasodilators may affect vessel tone and the state
of the blood nerve barrier.1 In this study, degranulated mast cells (as a percentage of total mast cells)
were present in highest numbers in eyes with recent
trauma (44.7 ± 14.8%, compared with 4.7 ± 1.9% for
normal optic nerves). This probably reflects a tendency for degranulation to occur as a result of mechanical factors. However, the histochemical techniques used are relatively insensitive to assessing degranulation, because cells that are devoid of granules
are not stained. This would systematically underestimate degranulation, and make difficult the correlation
of percentage mast cell degranulation with pathological processes resulting from release of mast cell mediators. Other techniques, such as immunohistochemical
detection of surface immunoglobulin E receptors,
may prove to be more sensitive in detecting degranulated mast cells.
Although in the current study most mast cells were
located in the optic nerve meninges, they were also
present in the parenchyma in certain pathological
conditions. Mast cells are generally considered to be
sessile, but they can be locally recruited in certain experimental and developmental situations. For example, we have shown that both the peripheral and central nervous systems of congenitally mast-cell-deficient strains of mice can be reconstituted with mast
cells by bone marrow transplant.3 In the current study,
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Investigative Ophthalmology & Visual Science, October 1993, Vol. 34, No. 11
FIGURE 7. Extracellular granules surrounding a typically appearing mast cell. The optic nerve was obtained from an
enucleated eye of a 36-year-old woman with neovascular
glaucoma from proliferative diabetic retinopathy. Partially
degranulated cells were considered degranulated for the
purpose of this study, recognizing that a completely degranulated cell would not be seen because of the absence of granules (chloroacetate esterase; original magnification X 315).
patients with vascular disorders, especially central retinal vein occlusion and branch retinal vein occlusion,
had a particularly high concentration of mast cells
(595.8 ± 140.I/mm3), which was almost as high as
those with inflammatory conditions (676.2 ± 210.6/
mm3). In three patients with neovascular complications of vessel occlusion and trauma, and one with
end-stage glaucoma, mast cells were found in the parenchyma. Mast cells are closely associated with blood
vessels, so it is not surprising that neovascularization
associated with those or other disorders may also recruit mast cells. It has been shown that mast cells migrate during normal development from pia mater
along blood vessels to perivascular locations within the
thalamus.13 Alternatively, nerve growth factor, which
increases numbers of mast cells, may be responsible
for locating foci of mast cells near vessels.1415 It is
possible that the pathological conditions occurring in
vascular occlusive or other diseases may result in a
recapitulation of these ontogenetic processes.
The role of mast cells in the optic nerve is unclear.
Although they may play a role in regulating vascular
permeability, diameter, and immunity, there is little
evidence for this within the optic nerve, nor the rest of
the central nervous system. Mast cells in and around
the optic nerve may have different functional implications from those found elsewhere in the central nervous system. In the brain, the surface to volume ratio
of meninges to parenchyma is clearly much lower, and
the average distance of cerebral tissue from meningeal
surface is much greater than in the optic nerve, where
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8. Acidified toluidine blue staining of section of an
optic nerve from a 7-month-old boy with retinoblastoma.
The mast cells are distinguished by their purple color, a metachromatic reaction of the stain with the mast cell granule
contents. No retinoblastoma was seen within any optic nerve
sections (original magnification X 50).
FIGURE
the distance between an optic nerve axon or glial cell
and the dura or arachnoid is never more than approximately 1.5 to 2 mm (half of the myelinated optic nerve
diameter of 3 to 4 mm). Therefore, the physiological
and pathological significance of mast cells in the optic
nerve is likely to be greater than in the brain. For example, localized tissue edema from mast cell degranulation may affect the more peripheral optic nerve parenchyma, possibly interfering with axonal action potential conduction. Mast cells that are aberrantly within
the nerve parenchyma may also affect nerve conduction, either by the same mechanism, or through the
direct action of granule contents on nearby astrocytes.16 The reason for the presence of mast cells in
this location is unclear; we have shown that mast cells
survive on cultured astrocyte monolayers,17 which suggests that optic nerve astrocytes may support mast cell
survival there. Finally, evidence from work with experimental demyelinating disorders suggests that mast
cells may play a role in autoimmunity within the nervous system.3"5'18-19 It is possible that mast cells located
in the meninges of the optic nerve, and within the
optic nerve parenchyma itself in patients with certain
ocular disorders, may be pathogenically significant in
certain optic nerve allergic and inflammatory conditions, and could be investigated by manipulating mast
cell activation in experimental models of optic neuritis.
Key Words
mast cell, optic nerve, meninges, chloroacetate esterase, optic neuritis
Mast Cells in Human Optic Nerve
3153
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