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Experimental Glaucoma and Cell Size, Density, and
Number in the Primate Lateral Geniculate Nucleus
Arthur J. Weber,1 Hao Chen,1 William C. Hubbard,2 and Paul L. Kaufman2
PURPOSE. To examine the effects that elevated intraocular pressure (IOP), a glaucoma risk factor, has
on the size, density, and number of neurons in the primate lateral geniculate nucleus (LGN).
METHODS. The monkey model of experimental glaucoma was combined with standard histologic
staining and analysis techniques. Fourteen animals were examined.
RESULTS. Mean IOPs higher than 40 mm Hg for 2.5, 4, 8, and 24 weeks resulted in reductions of 10%
to 58% in the cross-sectional areas of LGN neurons receiving input from the glaucomatous eye.
Reductions for animals with lower mean IOPs (37 and 28 mm Hg) for 16 and 27 weeks were 16%
and 30%, respectively. Neurons receiving input from the normal eye also were reduced in size
(4 –26%). No differential effect in cell size was seen for magnocellular versus parvocellular neurons.
Elevation of IOP resulted in an increase in cell density in all layers of the LGN. The increase was
approximately two times greater in parvocellular (59%) than magnocellular (31%) layers. When
corrected for volumetric shrinkage of the LGN, the estimated loss of neurons was approximately
four times greater in the magnocellular than parvocellular layers (38% versus 10%).
CONCLUSIONS. Elevation of IOP affects the size, density, and number of neurons in the LGN, and the
volume of the nucleus itself. Although higher mean pressures (more than 40 mm Hg) reduce the
period during which these changes occur, comparable damage can be achieved by even moderate
(28 –37 mm Hg) levels of elevated IOP. On the basis of cell loss, elevation of IOP appears to have
a more profound degenerative effect on the magnocellular than on the parvocellular regions of the
LGN. (Invest Ophthalmol Vis Sci. 2000;41:1370 –1379)
I
n many vertebrates, the transfer of visual information from
the eye to visual cortex involves the relay of signals
through the dorsal lateral geniculate nucleus of the thalamus. In primates, the LGN comprises six distinct layers of
neurons, each receiving input from a single eye. The two
ventral layers contain relatively large neurons and are referred
to as the magnocellular (M) layers, whereas the four dorsal
layers contain smaller neurons, and are identified as the parvocellular (P) layers.1 Although visual information leaves the eye
in several parallel streams, the retinogeniculate projection in
primates typically is described as consisting of only two major
pathways, the M-pathway representing the projection through
the magnocellular layers and the P-pathway representing the
projection through the parvocellular layers. Functionally, the
M-pathway is thought to be concerned primarily with stimulus
movement and form, whereas the P-pathway plays a role in the
analysis of detail and color vision.2–5
In the visual system, as in other parts of the brain, neurons
depend on connections with other neurons for proper function and survival. Trauma or disease that affects cells in one
area typically also has a degenerative effect on neurons in
associated areas.6 – 8 In primary open-angle glaucoma, a disease
often characterized by elevated IOP, the primary site of damage appears to be the optic nerve in the region of the lamina
cribrosa.9 Because the axons of retinal ganglion cells form the
optic nerve, one consequence of glaucoma is the retrograde
degeneration of ganglion cells within the retina itself. Although
the degenerative effects that elevated IOP has on the optic
nerve10,11 and retina12–14 are well known, few data are available concerning transynaptic changes within the LGN.15–17 To
examine these changes, we compared the size, density, and
number of neurons in the M- and P-layers of normal monkeys
and monkeys with experimental glaucoma.
MATERIALS
From the 1Department of Physiology, Neuroscience Program, and
Center for Clinical Neuroscience and Ophthalmology, Michigan State
University, East Lansing; and the 2Department of Ophthalmology and
Visual Sciences, University of Wisconsin, Madison.
Supported in part by Alcon Laboratories, Fort Worth, Texas
(AJW), American Health Assistance Foundation (AJW), and National
Institutes Health of Grants EY-11159 (AJW), EY-02698 (PLK), and
RR-00167 to the Wisconsin Regional Primate Research Center.
Submitted for publication September 1, 1999; revised December
7, 1999; accepted December 16, 1999.
Commercial relationships policy: N.
Corresponding author: Arthur J. Weber, Department of Physiology, B-512 West Fee Hall, Michigan State University, East Lansing, MI
48824. [email protected]
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AND
METHODS
Subjects and Procedures
Fourteen monkeys (13 rhesus, Macaca mulatta; 1 cynomolgus, Macaca fascicularis), of both sexes, aged 6 to 18 years,
were used in this study (Table 1). All had clinically normalappearing eyes (slit lamp biomicroscopy, gonioscopy [Zeiss,
Thornwood, NY], and stereoscopic funduscopy by fundus
camera [Zeiss]), and all had baseline IOPs, measured by tonometer under ketamine HCl anesthesia (Goldman18 [Goldman,
Haag-Streit, Köniz, Switzerland] or Tono-pen XL; Mentor O&O,
Norwell, MA), below 21 mm Hg (normal IOP for ketamineInvestigative Ophthalmology & Visual Science, May 2000, Vol. 41, No. 6
Copyright © Association for Research in Vision and Ophthalmology
Glaucoma and the Primate LGN
IOVS, May 2000, Vol. 41, No. 6
1371
TABLE 1. Summary of Animals Studied
Animal
Sex
Age
(y)
Mean IOP*
(mm Hg)
M5559
M5562
M81102
AE96
AA02
M86005
M85128
M87035
M78075
M88081
M80142
G-1†
AF02
M78002
F
F
M
F
F
M
M
F
M
M
M
F
M
F
7
7
11
15
18
8
9
7
14
6
14
6
8
17
16
18
21/46 ⫾ 17
19/44 ⫾ 21
18/62 ⫾ 9
18/46 ⫾ 9
20/52 ⫾ 9
18/44 ⫾ 12
18/42 ⫾ 17
19/37 ⫾ 10
19/36 ⫾ 9
20/49 ⫾ 10
16/29 ⫾ 5
17/27 ⫾ 3
Peak IOP
(mm Hg)
Duration
(wk)
Cup/Disc and
Initial/Final Ratios
62
58
72
52
68
65
62
52
61
62
42
32
Normal
Normal
2.5
2.5
4
4
8
8
8
16
16
24
27
27
0.2
0.2
0.2/0.9
0.3/0.9
0.3/0.9
0.3/0.8
0.3/1.0
0.4/1.0
0.3/0.8
0.3/0.8
0.3/0.6
0.3/1.0
0.3/0.6
0.2/0.4
* Values are presented as baseline IOP/mean elevated IOP ⫾ 1 SD.
† Cynomolgus.
anesthetized monkeys, 10 –20 mm Hg). Topical proparacaine
HCl (Alcaine 0.5%; Alcon Laboratories, Fort Worth, TX) was
used for all procedures involving contact with the cornea, and
dilation of the pupils for fundus photography was achieved
with 2.5% phenylephrine HCl (Mydfrin; Alcon) and 1% tropicamide HCl (Mydriacyl; Alcon). All procedures were approved
by the Animal Care Committees of the University of WisconsinMadison and Michigan State University, and all adhered to the
ARVO Statement for the Use of Animals in Ophthalmic and
Vision Research.
Chronic elevation of IOP was induced by laser scarification of the trabecular meshwork.19,20 Each animal first was
anesthetized with ketamine HCl (10 mg/kg intramuscularly,
supplemented with 5 mg/kg intramuscularly as needed) and
sedated with diazepam (1 mg/kg intramuscularly). A standard
clinical argon laser (model 900; Coherent Radiation, Palo Alto,
CA) and slit lamp delivery system then was used to produce a
series of focal lesions in the trabecular meshwork in one eye
(50 –250 spots, 50-␮m spot diameter, 1–1.5 W, 0.5 second
duration). IOP was monitored for 2 to 3 weeks after treatment,
and if not consistently 25 mm Hg or more, additional laser
treatments were performed until stable ocular hypertension
was achieved. The opposite eye served as the normal control.
At least once per week after laser treatment, the normal
and treated eyes were examined with the slit lamp, and corneal
clarity, anterior chamber cells, and flare were noted. After
measurement of IOP, the anterior chamber angle and optic
nerve head were examined with a goniolens (Zeiss; or model
OG3M-13; Ocular Instruments, Inc., San Jose, CA). Fundus
photographs were obtained every 3 to 4 weeks, depending on
the clinical appearance of the optic disc compared with its
appearance in previous eye examinations.
After 2.5 to 27 weeks of elevated IOP, the animals were
anesthetized deeply with ketamine HCl (15 mg/kg, intramuscularly), followed by intravenous pentobarbital sodium (35
mg/kg). The eyes then were removed for intracellular analysis,14 and the animals were perfused transcardially with 0.5 l of
0.9% saline followed by 1 to 2 l of 10% formol-saline solution.
The brains were removed and postfixed for 4 to 6 weeks with
several changes of fresh fixative.
Tissue Processing and Correction for Shrinkage
After fixation, each brain was blocked in the frontal plane,
dehydrated in a series of graded alcohols, and embedded in
celloidin. The LGNs then were sectioned serially at 25 ␮m in
the coronal plane, and every fifth section was stained with
thionine, mounted on a glass slide, and coverslipped for histologic analysis. Care was taken to standardize the dehydration
and celloidin processing times for each brain.
Linear tissue shrinkage, assumed to be uniform, was determined by inserting a pair of fine tungsten wires into each
block of LGN tissue and carefully measuring the distance between the wires, at the level of the tissue, before and after the
dehydration and celloidin embedding processes. The percentage linear shrinkage for each block of tissue then was derived
from the formula SL ⫽ 100 ⫺ (AP/BP ⫻ 100), where BP and AP
represent the before and after processing distances between
the tungsten wires, respectively. On the basis of linear shrinkage (SL), it then was possible to derive correction factors for
both areal and volumetric shrinkage. The correction factor for
areal shrinkage (ACF ⫽ 1/(1 ⫺ SL)2, when multiplied by the
measured area of each neuron or LGN region (M or P), then
yielded the true area of that structure. The correction factor for
volumetric shrinkage was derived from the measurement of
linear shrinkage (SL) using the formula: VCF ⫽ 1/(1 ⫺ SL)3.
Cell Size
The effects of elevated IOP on cell size were studied by measuring at ⫻1000 the cross-sectional areas of approximately
2400 geniculate neurons in each animal. Because glaucomatous visual field loss is most common within the central
30°,21all neurons were sampled from a rostral– caudal location
of the nucleus that corresponded to a visual field representation of approximately 10 to 15° azimuth.22–24 In addition,
because retinal ganglion cell loss in glaucoma can be diffuse,
LGN regions representing retinal input from superior retina
(nasal LGN), inferior retina (temporal LGN), nasal retina (contralateral LGN), and temporal retina (ipsilateral LGN) were
examined (Fig. 1). Cell samples (approximately 100 neurons/
layer) were taken from the full width of each lamina and
included only those neurons with clearly visible nucleoli. Measurements of soma area were obtained from the cell drawings
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Weber et al.
IOVS, May 2000, Vol. 41, No. 6
(model C5985 chilled CCD; Hamamatsu, Bridgewater, NJ) and
image analysis software (Image-Pro Plus; Media Cybernetics,
Silver Spring, MD). The laminar area of the M and P region
included in each section was obtained by measuring the total
area of each region and subtracting that portion occupied by
interlaminar space. Total laminar volumes for the M and P
regions of each animal were derived by summing the measured
areas, multiplying by section thickness (25 ␮m), and correcting
for sample size (every fifth section) and volumetric tissue
shrinkage.
Statistical Comparisons
FIGURE 1. Primate retinogeniculate pathway showing the regions of
the LGN examined and the approximate locations of their retinal
inputs. Layers 1 and 2 are the M-layers and layers 3 through 6 are the
P-layers. Ganglion cells in nasal retina project to the contralateral LGN,
whereas those in temporal retina project ipsilaterally. The nasal region
of the LGN receives its afferent input from ganglion cells in superior
retina (B, C), and the temporal region of the LGN is innervated by
ganglion cells located in inferior retina (A, D).
using a digitizing tablet and commercial software (SigmaScan;
Jandel Scientific, Corte Madera, CA). All cell area measurements were corrected for tissue shrinkage.
Cell Density and Number
Cell density in the M- and P-layers was estimated by counting
with a grid eyepiece reticle and ⫻50 oil immersion objective all
neurons displaying clear nucleoli that were located within a
sample volume that was 130 ⫻ 130 ⫻ 25 ␮m (section thickness). Cell counts were derived from three regions per layer in
each of three adjacent LGN sample sections (every fifth section), for nine sample regions per LGN layer. The rostral–
caudal and medial–lateral location of each sample region corresponded with the approximate LGN location from which cell
size measurements were obtained. All cell counts were corrected according to the method of Abercrombie,25 using the
diameter of the nucleolus (approximately 3 ␮m) as the object
thickness. All sample areas were corrected for volumetric
shrinkage as described. Estimates of total cell number for the M
and P regions of normal and glaucomatous animals were derived by multiplying the corrected sample densities (neurons/
cubic millimeter) by their respective LGN volume measurements (in cubic millimeters). It is important to note that our
measurements of LGN cell density and number for normal
animals agree closely with similar estimates made using comparable histologic techniques and both the Abercrombie25 and
optical dissector stereologic methods.23
LGN Volume
LGN volume was determined by capturing digital images of
each LGN sample section using a high-resolution video camera
All data are presented as means ⫾ 1 SD. Differences in mean
soma size and cell density were compared using two-way
analysis of variance (ANOVA) statistical methods (SPSS, Chicago, IL) combined with the Bonferroni adjustment for multiple comparisons (NCSS 2000, Kaysville, UT). Paired comparisons of LGN cell size distributions were made using the
Kolmogorov–Smirnov test for two independent samples
(SPSS). Differences in LGN volume and cell number were
compared using one-way ANOVA, followed by the Dunnett
test for multiple comparisons (SPSS). Correlation and partial
correlation analyses (SPSS) were used to compare the relations
between mean, peak, and duration of elevated IOP; cell size,
density, and number; and LGN volume. In all cases, P ⫽ 0.05
was used as the level of significance.
RESULTS
Qualitative Observations
Figure 2 shows examples of the cellular organization of the
normal primate LGN (Fig. 2A) and after different periods of
elevated IOP (Figs, 2B, 2C, 2D). The sections are presented in
the coronal plane, with the nasal region of the nucleus to the
left. The two ventral layers (Fig. 2A, layers 1, 2) are the Mlayers, and the four dorsal layers (Fig. 2A, layers 3 through 6)
are the P-layers. The LGN shown in Figure 2B is from animal
M81102 (2.5 weeks, 46 mm Hg). Although at the time of death
the optic nerve head was deeply cupped and the temporal
region of the disc was pale, the LGN did not appear abnormal
(cf., Fig. 2A). By contrast, the LGN in Figure 2C (animal
M85128: 8 weeks, 52 mm Hg), displays clear cellular differences between those layers receiving input from the glaucomatous eye (affected layers: 2, 3, and 5) and those receiving
input from the normal eye (unaffected layers: 1, 4, and 6).
Neurons in the affected layers appear to be smaller and less
well stained than those in the unaffected layers, and this cellular difference becomes more pronounced after 6 months of
relatively high mean IOP (Fig. 2D). At the time of death, the
glaucomatous eye of this animal (G-1: 49 mm Hg) was deeply
cupped and uniformly pale, and intracellular analysis of the
retina14 indicated that few ganglion cells remained. For animals
with 4 weeks of elevated IOP, the LGN of the animal with the
lower mean IOP (M86005: 46 mm Hg) resembled that of the
2.5-week animals (Fig. 2B), whereas the LGN from the animal
with the higher mean IOP (AA02: 62 mm Hg) more closely
resembled the LGN of the 8-week animal shown in Figure 2C.
The LGNs from animals that had 16 and 27 weeks of elevated
IOP, but lower mean levels (37 and 28 mm Hg, respectively),
most closely resembled the LGNs of the 2.5- and 8-week animals shown in Figures 2B and 2C.
IOVS, May 2000, Vol. 41, No. 6
Glaucoma and the Primate LGN
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FIGURE 2. Photomicrographs of cresyl violet–stained coronal sections
from the right LGN of a normal animal (A) and animals that had the
pressure in one eye elevated for 2.5
(B), 8 (C), and 24 weeks (D). In all
cases, the nasal region of the nucleus
is to the left, and the temporal region
is to the right. Layers 1, 4, and 6
receive retinal input from the normal
contralateral eye, whereas layers 2, 3,
and 5 are innervated by the glaucomatous ipsilateral eye. Prolonged elevation of IOP resulted in a decrease
in the size and Nissl substance within
neurons receiving input from the
glaucomatous eye, resulting in their
pale appearance. Scale bar, 500 ␮m.
Quantitative Observations
Cell Size. Because chronic elevation of IOP may not affect
ganglion cells in all areas of the retina equally, we examined
LGN regions receiving input from the superior, inferior, nasal,
and temporal hemiretinas of each eye (Fig. 1). The results of
these regional comparisons, in animals with mean IOPs more
than 40 mm Hg, are shown in Figure 3. For neurons receiving
input from superior versus inferior retina (Fig. 3A), no selective
difference was seen within either the M- or P-layers of the LGN.
Neurons receiving input from both hemiretinas showed approximately an 8% decrease in soma size after 2.5 weeks of
elevated pressure. After 4 to 8 weeks, the mean soma size for
neurons in both the M and P regions was only 75% of normal.
After 6 months of elevated IOP, neurons in the M-layers had
decreased in size approximately 58% (from 490 to 208 ␮m2),
FIGURE 3. Comparison of the change
in mean soma size versus duration of
elevated IOP for M- and P-cells receiving input from ganglion cells located in
either the superior versus inferior (A)
or nasal versus temporal (B) regions of
the retina. Comparable decreases in
mean soma size were measured in all
retinal target regions of the LGN (*P ⬍
0.05 versus normal; 2-way ANOVA
with Bonferroni adjustment).
whereas neurons in the parvocellular layers had decreased
approximately 59% (from 289 to 118 ␮m2). The 16-week animals (mean IOP: 37 mm Hg) showed approximately a 16%
decrease in LGN cell size in both the M- and P-layers, whereas
those with 27 weeks of elevated IOP (mean IOP: 28 mm Hg)
showed a decrease of approximately 32%.
Cell size comparisons for LGN neurons receiving input
from nasal versus temporal retina also showed no differential
effect (Fig. 3B). After 2.5 weeks of elevated IOP, neurons in
both the M- and P-layers were approximately 8% smaller than
normal. By 4 weeks the mean soma size of M-cells receiving
input from either hemiretina had decreased from 492 to 340
␮m2 (31%). Over this same period, parvocellular neurons
showed a decrease in area of approximately 26% (from 289 to
215 ␮m2). Although little additional change in soma size oc-
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Weber et al.
curred for LGN neurons located in either region between 4 and
8 weeks, cells in both the M- and P-layers underwent an
additional 10% to 15% decrease in soma size between 8 and 24
weeks, reaching final mean values of 230 and 130 ␮m2, respectively. Nasal–temporal differences in soma size for M- and
P-cells from animals with 16 and 27 weeks of elevated IOP
were comparable with those described for neurons receiving
input from superior versus inferior retina: approximately a 16%
decrease in both M- and P-cells after 16 weeks of elevated IOP
and approximately a 28% decrease in the size of M-cells and
34% decrease in the size of P-cells with 27 weeks of elevated
IOP.
Chronic elevation of IOP also resulted in a decrease in the
mean soma sizes of LGN neurons receiving input from the
normal eye (Fig. 4). The percentage difference in soma size for
both M- and P-cells within the affected versus unaffected layers
was small after 2.5 to 4 weeks of elevated IOP (3.6 –5.3%). This
difference increased to approximately 14% by 8 weeks, and to
48% after 24 weeks. Animals with 16 weeks of elevated IOP
also showed decreased mean soma sizes in both the affected
and unaffected layers. Neurons in the unaffected M- and Players showed an approximately 12% decrease in mean soma
size, and those in the affected layers were 16% smaller than
normal. Despite mean IOPs of only approximately 28 mm Hg,
and their showing little change in cup-to-disc ratio (Table 1),
the animals with 27 weeks of elevated IOP showed approximately a 26% decrease in M-cell size and a 34% decrease in
P-cell size for both affected and unaffected layers.
Figure 5 compares the size distributions of neurons in the
magnocellular (Figs. 5A, 5B) and parvocellular (Figs. 5C, 5D)
regions of the normal and glaucomatous LGN. The upper curve
in each plot represents the size distribution of neurons located
in the right temporal region of the normal LGN, whereas the
lower curves represent the size distributions of neurons mea-
FIGURE 4. Comparison of pressure-induced changes in mean soma
size for neurons located in the affected and unaffected layers of the
LGN. Neurons in the affected layers receive direct synaptic input from
axons of ganglion cells located in the glaucomatous eye, whereas those
in the unaffected layers receive their input from ganglion cells in the
normal eye. Neurons in both regions showed changes in soma size as
a result of chronic elevation of IOP (*P ⬍ 0.05 versus normal; 2-way
ANOVA with Bonferroni adjustment).
IOVS, May 2000, Vol. 41, No. 6
sured in matched regions of animals with 2.5, 4, 8, and 24
weeks of elevated IOP. In the normal LGN, M-cells (Figs. 5A,
5B) ranged in size from approximately 121 to 799 ␮m2, with a
mean soma area of 474 ␮m2. As expected, normal P-cells (Figs.
5C, 5D) were smaller, ranging in size from approximately 108
to 525 ␮m2, with a mean soma area of 290 ␮m2. After 2.5 to 24
weeks of elevated IOP, neurons in the unaffected M-layers
were smaller than normal, averaging only approximately 400
␮m2. Although this decrease resulted in a small but significant
leftward shift in the cell size distributions for the 4-, 8-, and
24-week animals, the overall range of cell sizes for neurons in
the normal and unaffected M-layers was similar (Fig. 5A: normal, 121 to 799 ␮m2; unaffected, 119 to 802 ␮m2). The shift
toward smaller soma sizes was more pronounced in the affected M-layers, where both the overall mean and range of
soma sizes were significantly different from normal (Fig. 5B:
mean, 324 versus 400 ␮m2; range, 130 – 680 ␮m2 versus 119 –
802 ␮m2). The unaffected P-layers (Fig. 5C) also showed a
modest but significant shift toward smaller soma sizes in both
mean cell area (229 versus 280 ␮m2) and the overall range of
cell sizes (108 – 472 ␮m2 versus 126 –525 ␮m2). Similarly, the
size distributions for neurons in the affected P-layers (Fig. 5D)
were significantly different from those for neurons in the
matched layers of normal animals. Neurons in the affected
layers ranged in size from 48 to 460 ␮m2 (mean, 196 ␮m2),
compared with a range of 108 to 472 ␮m2 (mean, 280 ␮m2) for
neurons in the normal layers. In all cases, LGN neurons from
animals with high mean pressures (approximately 49 mm Hg)
and long durations (24 weeks) of elevated IOP were affected
most severely.
Cell Density. Neuronal densities were derived from cell
counts made in approximately the same regions of the LGN
used to compare differences in cell size. In all animals, cell
densities for both the affected and unaffected regions of the Mand P-layers were obtained from measurements made in the
right LGN (Fig. 6). To reduce intralaminar variability, cell density measurements for normal animals were obtained only from
LGN layers that corresponded with the layers examined in the
experimental animals (e.g., animals with glaucomatous right
eyes had the cell densities in the affected layers of the right
LGN [layers 2, 3, and 5] compared only with the density of
neurons measured in layers 2, 3 and 5 of normal right LGNs).
Figure 6 shows that chronic elevation of IOP, regardless of
the magnitude, resulted in an increase in the density of neurons
in both the M- and P-layers. The percentage change in cell
density was greatest in the unaffected layers (M, 38%; P, 70%).
The increase in cell density for the unaffected layers of both
regions was significantly different from normal in animals with
8 weeks or more of elevated IOP. No significant difference was
found in changes in cell density in the affected layers, despite
a 45% increase in the P-layers. Changes in cell density in the
M-layers was more modest, showing a peak increase of 26%
after 8 weeks but increases of only 22% and 9% after 16 and 27
weeks, respectively. In comparing the affected and unaffected
layers, only the density of neurons in the affected M-layers of
animals with 27 weeks of elevated pressure was found to be
significantly less than the density of neurons in the unaffected
layers.
LGN Volume. Figure 7 shows the effects that different
levels and durations of elevated IOP have on LGN volume. As
indicated in the Materials and Methods section, these data
represent the total laminar volume (affected and unaffected
IOVS, May 2000, Vol. 41, No. 6
Glaucoma and the Primate LGN
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FIGURE 5. Three-point smoothed histograms showing the size distributions of M- (A, B) and P-cells (C, D) in the normal (upper traces) and
glaucomatous (lower traces) LGNs of animals after different durations of elevated IOP. Left: normal versus unaffected layers; right: normal versus
affected layers. For all comparisons versus normal (Kolmogorov–Smirnov test; P ⬍ 0.001, except 2.5-week unaffected magnocellular, P ⫽ 0.376).
layers) for each region. This served to minimize errors in
identifying affected versus unaffected layers in regions of the
LGN containing laminar discontinuities (e.g., Fig. 2C). As with
cell density, all volumetric data were derived from the right
LGN of each animal. However, comparable measurements
from the left LGN of four animals (normal, 8, 16, and 27 weeks)
indicated no left–right bias.
For normal animals, total laminar volume was found to be
approximately 68.2 mm3 (M, 56.4 mm3; P, 11.8 mm3). Both
regions of the nucleus showed significant decreases in laminar
volume after as little as 2.5 weeks of elevated IOP. The volume
of the P region decreased approximately 23%, to 43.4 mm3,
whereas the volume of the M region decreased 35%, to 7.7
mm3. Although 4 weeks yielded little additional change in
volume, both regions decreased to approximately 55% of normal by 8 weeks. A similar magnitude of change was seen in
both the M- and P-layers of the animals with 16 and 27 weeks
of elevated IOP, despite their lower mean pressures.
Neuronal Number. Estimates of total cell number in the
LGNs of normal and glaucomatous animals were determined
from the cell density and LGN volume measurements, after
corrections for tissue shrinkage (Fig. 8). For normal animals,
the total number of neurons in the LGN was estimated to be
approximately 1.27 million, with 10.3% located in the M-layers
and 89.7% in the P-layers. Compared with matched normal
layers, the affected M-layers of glaucomatous animals showed a
significant decrease (38%) in the number of surviving neurons
for each period studied, whereas the P-layers did not (10%
decrease).
Relation of IOP to Changes in LGN Morphology. Correlation and partial correlation analyses were used to analyze
the relations between mean, peak, and duration of elevated
IOP, and glaucoma-related changes in LGN cell size, density,
and number, as well as LGN volume (Table 2; ⫹ indicates P ⬍
0.05). In the M-layers, correlations were found between mean
and peak IOP and decreases in cell size, number, and LGN
volume. No correlation was seen for duration of IOP versus cell
size, or cell density versus any component of IOP. In the
P-layers, correlations were found between mean, peak, and
duration of elevated IOP and the decrease in cell size and LGN
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Weber et al.
IOVS, May 2000, Vol. 41, No. 6
FIGURE 8. Histograms showing estimates of the mean number of
neurons in the normal and affected M (left) and P (right) regions of the
glaucomatous LGN after different durations of elevated IOP (*P ⬍ 0.05
versus normal; 1-way ANOVA with Dunnett test).
FIGURE 6. Comparison of the change in cell density in the affected
and unaffected M- and P-layers of the LGN after different durations and
mean levels of elevated IOP (*P ⬍ 0.05 versus normal; 2-way ANOVA
with Bonferroni adjustment).
volume, as well as the increase in cell density within this
region. No correlation was seen between any component of
IOP and the decrease in P-cell number.
DISCUSSION
We used the monkey model of glaucoma and standard histologic techniques to examine the degenerative effects that
chronic elevation of IOP has on the cytoarchitecture of the
primate LGN. The results show that elevation of IOP had a
profound effect on the size, density, and number of neurons in
the LGN, as well as the laminar volume of the nucleus itself. In
FIGURE 7. Histograms showing the change in laminar volume for the
M- and P-layers of the LGN as a result of different levels and durations
of elevated IOP. For each region, total laminar volume was derived by
combining the volumes of the affected and unaffected layers, and
excluding intralaminar volume (*P ⬍ 0.05 versus normal; 1-way
ANOVA with Dunnett test).
addition, they show that these cellular changes were not restricted to LGN layers receiving input from the glaucomatous
eye, but also were present, albeit to a lesser degree, in laminae
innervated by the normal eye.
The cell size data reveal two important relationships between elevation of IOP and LGN degeneration. First, they show
that longer durations of elevated IOP, even at low to moderate
levels, resulted in larger reductions in the soma sizes of both Mand P-cells. Second, they indicate that higher mean levels of
IOP shortened the time during which the cellular changes
occur. Animals with 2.5 weeks of elevated pressure at mean
levels of approximately 45 mm Hg showed decreases in mean
soma size that were comparable with animals with 16 weeks of
elevated IOP and mean levels of approximately 36 mm Hg.
Similarly, animals with 8 weeks of elevated IOP (approximately
46 mm Hg) displayed reductions in mean soma size that were
comparable with those with 27 weeks of elevated IOP and
mean IOPs of approximately 28 mm Hg.
At each of the different periods studied, similar percentage decreases in mean cell area were measured in both the Mand P-layers. Although this suggests that IOP affects the sizes of
M- and P-cells equally, it is important to note that these data
reflect changes in the population mean and not necessarily
those of single neurons. This distinction is important, because
different populations of neurons may reach their final mean
sizes through completely different mechanisms (e.g., cell loss
versus shrinkage). The histograms in Figure 5 suggest that both
cell loss and shrinkage contribute to the decrease in mean
soma size within the M- and P-layers in glaucoma. They also
indicate that in both regions the largest neurons may be affected most severely. Both areas show an absence of very large
neurons, and both display modest increases in the percentage
of small to medium sized neurons, as would be expected if
larger neurons also were shrinking (cf., Figs. 5B, 5D). That cell
size, and not cell class, may be the main factor governing
glaucomatous changes in the LGN is consistent with similar
results in the primate retina.12,13 The leftward shift of the
lower ends of the parvocellular, but not magnocellular, distributions for animals with 4, 8, and 24 weeks of elevated IOP
suggests that general cell shrinkage might play a greater role in
determining final mean soma size in the P-layers, whereas cell
loss is the primary mechanism in the M-layers. This would be
Glaucoma and the Primate LGN
IOVS, May 2000, Vol. 41, No. 6
1377
TABLE 2. Correlation/Partial Correlation Analysis: IOP Versus Cell Morphology
Magnocellular
Mean IOP
Peak IOP
Duration
Parvocellular
Size
Density
Number
Volume
Size
Density
Number
Volume
⫹
⫹
⫺
⫺
⫺
⫺
⫹
⫹
⫹
⫹
⫹
⫹
⫹
⫹
⫹
⫹
⫹
⫹
⫺
⫺
⫺
⫹
⫹
⫹
⫹ P ⬍ 0.05.
consistent with our cell number estimates: We found approximately a four times greater loss of M-cells than P-cells in the
glaucomatous animals.
To date, only one study has examined cellular changes in
the primate LGN with glaucoma. Chaturvedi et al.15 used
human autopsy material to compare the density of neurons in
the magno- and parvocellular regions of normal patients and
patients with documented glaucoma. Based on a small (approximately 1 neuron/per square millimeter) but significant decrease in cell density in the M- but not P-layers, the authors
concluded that glaucoma has a preferential effect on the Mregion of the LGN. Our data also indicate that glaucoma has a
differential effect on M- versus P-cell density. However, they
show that chronic elevation of IOP results in an increase in
LGN cell density and that this increase in cell density is approximately two times greater in the P-layers than in the
M-layers (59% versus 31%). The large increase in P-cell density
most likely resulted from a significant decrease in laminar
volume, but not cell number, in this region of the nucleus,
whereas the modest increase in M-cell density probably reflected a complex balance between decreasing laminar volume
and cell loss (Figs. 7, 8). As expected, the largest increases in
cell density were measured in the unaffected M- and P-layers,
where cell loss was minimal.
The apparent contradiction between our cell density results and those of Chaturvedi et al.15 could be explained by
animal– human variation or differences in the duration of glaucoma for their patients (years) versus our animals (weeks). If
during the initial stages of the disease process, cell density is
influenced primarily by a rapid reduction in LGN volume due
to early axonal degeneration (described later), then the increases in cell density reported here would be expected. As the
disease progresses and cell loss becomes more prevalent, then
either no change or a decrease in cell density might be expected, depending on the relation between LGN volume and
cell number. This most closely reflects the results of Chaturvedi et al.15
In agreement with Ahmad and Spear,23 we found the total
laminar volume of the normal rhesus LGN to be approximately
68 mm3. After only 2.5 weeks of elevated IOP, the volume of
the P-layers decreased approximately 23%, whereas the Mlayers underwent a 35% reduction in size. Despite its smaller
percentage decrease, the actual reduction in P volume is approximately threefold greater than the reduction in M volume
(13 mm3 versus 4.2 mm3). Because P-cells undergo little
change in size or number during this period (Figs. 4, 8), this
initial decrease in P volume most likely reflects an early loss of
axonal material.26 The more pronounced change within the P
region might be due to several factors. First, because the P
region contains approximately eight times as many neurons,
there simply are many more retinal axons and terminal arbors
within this region. Second, because the axons of M-ganglion
cells enter the dorsal surface of the nucleus and traverse the
P-layers on their way to the ventral M region,27 atrophy of
these fibers also may contribute to a decrease in parvocellular
volume. Within the M-layers, the decrease in laminar volume
most likely involves a complex combination of axon degeneration, cell shrinkage, and neuronal cell loss (Figs. 4, 5, and 8).
One unexpected result of this study was the relatively
constant level of cell loss across animals with different durations of elevated IOP (Fig. 8). After 2.5 weeks of elevated
pressure, the affected M-layers show approximately a 38% loss
of neurons, and little change thereafter. A similar pattern was
seen for the P-layers; however, the onset of cell loss was
delayed approximately 1.5 weeks relative to the M-layers. One
possible explanation for this step-like pattern in LGN cell loss
is that each region contains subpopulations of neurons with
different sensitivities to optic nerve injury. Although the data in
Figure 8 may represent an initial loss of the most sensitive
neurons, longer durations of elevated IOP may be needed to
show the loss of more resistant neurons. That the M-layers of
the primate LGN may contain neurons with different sensitivities to axonal injury has been suggested previously,28 and
Golgi studies have described at least two types of relay cells in
this region of the nucleus.29
An obvious question is, why do LGN neurons degenerate
after damage to the optic nerve? In the case of retinal ganglion
cell death and glaucoma, pressure-induced damage to the optic
nerve has been shown to disrupt axonal transport,9,30,31 which
then results in a decrease in the amount of trophic material
ganglion cells obtain from their target neurons in the LGN.
Because elevation of IOP does not damage LGN neurons or
their connections with target neurons in visual cortex directly,
it seems likely that neurons in the glaucomatous LGN degenerate primarily because of a decrease in neuronal activity
within the retino-geniculo-cortical pathway. LGN neurons may
require a minimum level of activity to maintain connections
with visual cortex and obtain sufficient amounts of trophic
material for their survival. A reduction in neuronal activity may
also explain the cellular changes seen within the unaffected
layers, a phenomenon not restricted to glaucoma.8,32 Because
LGN neurons representing each eye innervate common targets
in visual cortex, a reduction in cortical activity that produces a
general decrease in cortical trophic levels would have an effect
on cells in all layers of the nucleus.
At present, there is good evidence that retinal ganglion
cells in the glaucomatous eye die by apoptosis.33,34 Although
similar studies have not been conducted in the LGN, transynaptic apoptotic cell death has been described in other systems.35,36 This does not preclude concomitant involvement of
1378
Weber et al.
necrotic cell death, however, because both necrosis and apoptosis have been demonstrated in the LGN after visual cortex
damage37 and in the retina after pressure-induced ischemia38,39
and optic nerve crush.40 Interestingly, neurons undergoing
each type of cell death appear to follow a different time course:
Neurons that die soon after the insult do so mainly by necrosis,
whereas those that die later undergo apoptosis.
Although we cannot address the functional significance of
our cellular changes, it is of interest to note that Smith et al.41
reported few abnormalities concerning the functional integrity
of LGN neurons or their retinal afferents in monkeys with
experimental glaucoma. This result is somewhat surprising,
considering the structural abnormalities of ganglion cells in the
glaucomatous eye14 and the transneuronal changes described
in this study. One possible explanation is that retinal ganglion
cells and LGN neurons are capable of maintaining much of
their functional integrity, despite significant changes in morphology. Current retinal studies are examining this possibility.
Although many questions remain, these data demonstrate
clearly that chronic elevation of IOP has a profound degenerative effect on the primate LGN, a major structure involved in
the integration and transfer of visual information. Although our
estimates of pressure-induced cell loss suggest that glaucoma
has a more pronounced effect on neurons in the M- than in the
P-pathway, perhaps the more important message is that in
strategies intended to mitigate glaucomatous neuropathy, treatment of the entire central visual pathway, and not the eye
alone should be considered.
Acknowledgments
The authors thank Todd Perkins and Michael Bueche for assisting with
clinical evaluations of the eyes; Elaine Bostad, Jane Walsh, Judy McMillan, and Charles Greenfeld for technical assistance; and Dan Houser
and Shelly Zimbric of the Wisconsin Regional Primate Research Center
for assistance with the animals.
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