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1 From the Department of Ophthalmology and Visual Science, University of Texas Medical School at Houston; and 2 College of Optometry, University of Houston.
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Abstract |
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METHODS. Monocular experimental glaucoma was induced in adult monkeys (Macaca mulatta and Macaca fascicularis) by laser application to the trabecular meshwork, increasing the intraocular pressure. After other experiments, the primary visual cortices were analyzed for functional excitation from surviving ganglion cells, as indicated by cytochrome oxidase histochemistry.
RESULTS. Cytochrome oxidase reactivity was uniformly reduced in blobs with input from the glaucomatous eye in a manner consistent with loss of known afferent inputs. The average size of glaucomatous blobs in layers 2 and 3 of V1 cortex was reduced by half.
CONCLUSIONS. Experimental glaucoma in monkeys reduces retinal input to the central nervous system, thereby reducing the metabolic drive to downstream targets, as indicated by the reduction in the size of cytochrome oxidase blobs in layers 2 and 3 of V1 cortex. The pattern of cytochrome oxidase loss within the blob was uniform, suggesting that all sources of afferent input to the blobs were affected by experimental glaucoma.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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CO is an essential mitochondrial membrane protein for brain oxidative metabolism, catalyzing the last step in the formation of adenosine triphosphate (ATP), the energy source for neuronal function.2 3 4 The brain tissue content of CO is inhomogeneously distributed4 5 6 and varies with the metabolic demand attendant with neuronal activation.7 8 9 10 11 12 In the afferent visual system of primates, histochemistry has shown CO reactivity to decrease monotonically with the duration of sensory visual deprivation by enucleation or by tetrodotoxin (TTX) blockade.13 14 Therefore, it is reasonable to expect CO reactivity to be at covariance with the level of activation of neurons in the monkey afferent visual system, including the blobs of superficial layers 2 and 3.
CO-rich blobs are oft described, and now well-known, metabolic features
of the superficial layers 2 and 3 of primary visual cortex. In most
primates, CO blobs are distributed in periodic rows directly above the
center of the eye-dominance columns (ODC) of input layer 4C and are
known to derive a major afferent input from the two laminar
subdivisions, layer 4C
and 4Cß, directly beneath. In turn, the
input to these two subdivisions comes from the two major ganglion cell
projection pathways, with the M-cell pathway (carrying primarily
retinal information gathered by the large parasol retinal ganglion
cells) projecting into layer 4C, and the P-cell pathway (carrying
retinal information gathered by the small- to medium-sized retinal
midget ganglion cells) projecting into layer 4Cß. In addition to
these cortical afferents, the CO blobs get a direct thalamic input from
the K-cell laminae of the lateral geniculate nucleus (carrying retinal
information gathered by small bistratified ganglion cells). Although
both layer 4C subdivisions send projections to superficial layers 2 and
3, the CO blobs are thought to receive a major input from layer
4C.15
Whereas the surrounding interblob areas get input
from both divisions of layer 4C, the input from 4Cß contributes far
more synaptic contacts.16
Because these studies have shown
that there are multiple afferent inputs to CO blobs, and because we and
Vickers et al.17
have shown that experimental glaucoma
reduces CO staining in the macaque afferent visual system and we have
shown a differential effect of experimental glaucoma on two of the
primary afferent inputs to the blobs,1
we next measured
changes in the size and metabolism of the CO blobs in these same
monkeys.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Figure 1 is presented to indicate the level of glaucomatous damage in the five monkeys whose visual brains were analyzed in this article. The figure shows the HVFM for four monkeys. Figure 1A is a map (OHT19) typical of the threshold for detecting a visual defect, whereas the HVFMs of Figs 1B 1C and 1D are from monkeys used in the present study, and show severe defects from experimental glaucoma (OHT18, OHT9, and OHT21, respectively). Two other animals (M. fascicularis AL540 and AL203) were treated similarly, but systematic treatment and behavioral data were not collected on them. The visual field defects shown in Figures 1B 1C and 1D are indicative of advanced glaucomatous damage and a severe loss of retinal ganglion cells. Although counts of the loss in ganglion cells in these experimental animals are in preparation and incomplete, it is clear from our comparable counts in other animals treated similarly (e.g., see Reference 19 Fig. 2 ) that all animals analyzed in this report had advanced glaucomatous retinal damage.
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Tissue Preparation
At the end of the behavioral or electrophysiological study, the
animals were killed by overdose (100 mg/kg) of pentobarbital sodium
(Nembutal; Abbott, Abbott Park, IL), and exsanguinated with 2 l of
saline followed by 2 l of a 2% paraformaldehyde–0.5%
glutaraldehyde fixative in phosphate buffer (pH 7.4). The brain and
optic nerves were removed and (in most cases) the visual cortices were
dissected and gently flattened on a glass slide. The tissue was
refrigerated overnight in the fixative before beginning a sucrose
dehydration gradient of 10%, 20%, and 30%. The visual cortices were
embedded in aluminum foil cups filled with TissueTek (Miles, Elkhart,
IN) and frozen by lowering into an acetone bath cooled by liquid
nitrogen. Tangential sections of 30-µm thickness were collected and
stained for the histochemical localization of CO according to the
protocol of Wong–Riley.2
The CO-stained sections were
mounted on gelatinized slides, dehydrated, and coverslipped (Permount;
Fisher Scientific, Fairlawn, NJ).
Optical Measurements
The CO-stained sections were homogeneously back illuminated, and
a digital image was captured at a 1200-pixel resolution with a camera
(DCM1; Polaroid, Cambridge, MA), which was linear with optical density,
with an R = 0.99. The density of the COR was scaled 0
to 255, where 0 = opacity and 255 = the incident light.
Measurements were taken before any filtering or contrast enhancement.
COR = I - T, where I was the incident light (nominally, a
value of 255), and T was the light transmitted through the tissue
containing the CO-reaction product. The value of COR from a blob
connected with the glaucomatous experimental eye
(CORG) was always compared in the same section
with the value of COR measured in the directly adjacent blob connected
with the normal companion eye (CORN). The ratio,
CORG/CORN, constituted the
primary data from which mean values and variances were calculated. The
mean COR and SD of a closed contour, drawn by eye to best outline the
blob, were recorded for each blob and compared with the values obtained
in the companion blob with primary input from the opposite eye. Thus,
the ratio of CORG/CORN was
formed. A minimum of 10 such ratios was then averaged for each tissue
location. Most often, the ratio
CORG/CORN was expressed as
a percentage reduction of COR, relative to that in the companion site
that had input from the normal eye.
For the relative size measurement, the numbers of pixels contained within the closed contour of the tracing of the CO blob image was compared. It is recognized that there is no generally accepted objective criterion for drawing the CO blob boundaries, because there is a characteristic decreasing COR gradient from the blob center toward the edge.14 Therefore, we present a statistical comparison of the subjective sizes of pairs of CO blobs, collected by a technician naive to the purposes of the study, and the results evaluated by the paired t-test.
The brain section image was processed using the University of Texas at San Antonio Image Tool 2.0 image analysis software. With the reticulated CO pattern characteristic of layer 4A as a reference, the perimeters of the CO blobs in layer 3 were traced by eye and the numbers of pixels within the enclosed perimeter taken as an index of the relative area of blobs with input from the normal and the glaucomatous eye. The average area and relative COR were measured for pairs of CO blobs, one from the normal left (ODC), paired with the adjacent CO blob from the ODC of the glaucomatous right eye. A minimum of 10 pairs was measured from the V1 cortices of each of five of the experimental monkeys. The primary data were imported into a spreadsheet (QuattroPro; Corel, Ottawa, Canada) for computation and graphing, whereas statistical comparisons were made using the paired t-test (SigmaStat; Jandel Scientific, San Rafael, CA).
To describe the distribution of relative COR within the blob, pairs of normal and companion glaucomatous blobs were scanned along a line orthogonal to the course of the eye dominance columns. Smoothing the resulting noisy curves was achieved by subjecting the data to a 3-point rolling average to generate a 48-point profile for 10 pairs of blobs.
To present a different and enhanced view of the density, size, and distribution of COR within the blobs, the following operations were performed on selected blob fields. First, the density range of the image was inverted so that the low-intensity blob COR density signal, represented by the dark COR blobs, became high-intensity values. Next, the pixel values in the holes representing the blood vessels were replaced by the average value of the immediately surrounding pixels. A surface plot was then made so that the pixel intensity was coded both by height of the surface and by color. An azimuth and elevation was chosen to optimize inspection of differences between rows of blobs. In practice, low-pass filtering was often performed to accentuate the major features over high-frequency variations.
To fill and replace the blood vessel holes, two different protocols were used with approximately equal success. In the first protocol, the holes were selected in the image management software (Photoshop; Adobe, San Jose, CA) by selecting that range of intensities. A mask was created corresponding to the holes. The remainder of the image without the holes was then blurred by several passes of a median filter. The calculation facility of the software program was then used to replace the masked area corresponding to the holes with corresponding portions of the blurred image. This replaced the white holes with a local average of the surrounding area. In the second protocol, a custom "zippering" routine was implemented in a statistical analysis software program (The MatLab; MathWorks, Natick, MA). By this means, an upper threshold was imposed that distinguished the high-intensity holes from the darker tissue. The routine then replaced pixels exceeding this threshold with the value of their nearest neighbor not exceeding the threshold. By this mechanism, the surrounding regions "flooded" the holes with their values. This procedure was more robust in dealing with a wide variety of tissues but sometimes replaced the holes with values somewhat lighter than the near background, probably because of light scattering near the holes.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Figure 3 shows the relative COR values from the six visual cortices of the individual experimental animals for blobs associated with input from the glaucomatous eye (G) with those blobs with input from the normal eye (N). The average relative reduction in COR in blobs associated with the glaucomatous right eye for all the monkeys was 9% (range, 2–14%. The average COR tended to be reduced in the blob associated with the glaucomatous eye, with four of the six comparisons being significant reductions (paired t-test; P < 0.01).
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Overall, the average COR data for the five experimental monkeys (Fig. 3) shows that there was a significant reduction in the COR between the blobs having input from the glaucomatous right eye (RE) compared with those blobs having input from the normal left eye (LE). The distribution of the loss in COR within the blob is illustrated in Figure 4A , showing the COR profiles of 10 blob pairs. The shape of curve of the means and SD for 10 glaucomatous blobs was comparable with that of the companion 10 normal blobs, differing only in the lower relative level of COR in the glaucomatous blobs throughout. This suggests a uniform loss in COR throughout the blob, which is borne out in Figure 4B , showing a linear regression fit to the 24 mean values from the center of the blob into the interblob space. The parallel COR data and the regression curves attest to a uniform loss in COR throughout the glaucomatous blob.
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In summary, as the afferent input from ganglion cells to the visual brain is reduced by experimental glaucoma, the CO blobs of the superficial layers of primary visual cortex become smaller in a manner consistent with a uniform loss of metabolism within the blob.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The effects of glaucoma on the superficial CO-rich blobs of the V1 cortex are interesting in that the pattern of loss in COR suggests a uniform loss throughout the blob. Trusk et al.14 and Edwards et al.20 have characterized the density and pattern of change in COR within the normal CO blob, showing that COR is highest in the blob center, indicating that there is normally an underlying anisotropy in metabolic activity within the blob. Trusk et al.14 also showed that the COR reduced uniformly throughout the blob after deafferentation by enucleation, TTX blockade, and monocular eyelid suture conditions similar to that associated with deafferentation by glaucoma as we have shown here. Therefore, a uniform reduction in COR throughout the blob accounts for both the smaller blob size and the consistent COR profile in the blobs associated with the glaucomatous eye reported here.
Because the CO blob gets multiple inputs, it is surprising that the effect of glaucoma was uniform throughout the blob. Because we have shown1 that both P- and M-cell LGN layers supplying signals to V1 are metabolically, but differentially, inactivated by glaucomatous blockade, it is worthwhile to consider the contribution of those other pathways having input to the CO blobs.
One obvious source of stimulation to the CO blobs of layers 2 and 3 is the direct thalamic input from the koniocellular (K)-cells from the extralaminar zones of the LGN.21 22 23 As the P- and M-cell input to the blobs weaken and the blob shrinks, the K-cell input could sustain elevated metabolism within the shrinking blob. However, because the K-cell itself gets a major afferent input from a third morphologic and functional class of retinal ganglion cell, the small bistratified ganglion cell23 24 (thought to carry blue–yellow color information; see recent review25 ), it too would probably be subject to the same deleterious effects of glaucoma. Although the K-cell receives other input (first from the superficial layers of the superior colliculus [SC], as well as from the parabigeminal nucleus [PG]), it is hard to see how these indirect afferent routes into the CO blobs could sustain metabolic activity, because both these sources are themselves dependent on retinal ganglion cell input. For example, the SC superficial layers get a large projection from V1, layer 5, which would be downstream of the impaired P-, M-, and K-cell geniculocortical projection just discussed. Moreover, it would be expected that advanced glaucoma would weaken the direct ganglion cell input to SC in a similar manner to the effects shown for the geniculocortical projection.
Moreover, if one uses the cell size argument in the sequence of impairment and death of ganglion cells in glaucoma26 (i.e., that it is the ganglion cells with the largest soma size that are first affected in glaucoma) the small bistratified ganglion cell supplying input to the K-cell would have no particular survival advantage, in that these ganglion cells have soma sizes intermediate to midget and parasol ganglion cells having input to the P- and M-cell LGN layers, respectively. Therefore, it is unlikely that K-cells would have any survival advantage in glaucoma.
Because it has been suggested that the collective input to the blobs
from the three LGN sources (layers 4C, 4Cß, and K-cell) represents
less than 20% of the synaptic connections within the CO
blob,25
most of the synaptic contacts within the blob must
come from intracortical sources. For example, if the mosaic of blobs
has some binocular interconnections (either excitatory or inhibitory)
the active blobs connected with the normal eye could affect stimulation
to the CO blob connected with the glaucomatous eye. What evidence is
there for such a pathway?
Several studies have shown that there are extensive intracortical
lateral projections (spreading axons with periodic terminations) within
the superficial layers of V1 of primates. Orthograde tracers have been
injected within the layer 3 CO blob, and the characteristics of the
lateral axonal spread described.27
Malach et
al.28
showed that biocytin injections within one
CO blob marked axonal spread that tended to contact adjacent blobs
connected with the same eye, but not to the blobs connected with the
opposite eye. However, using similar methods, Yoshioka et
al.29
have shown that these lateral projections spread
predominantly orthogonal to the course of the ODCs, where axonal
branches contact most of the adjacent CO blobs. These two studies
reinforce the original paper on the subject by Livingstone and
Hubel30
who used horseradish peroxidase to show that blobs
are connected with blobs, and nonblob areas are interconnected with
other nonblob areas. These studies agree that there is a statistical
preference of these lateral projections to link CO blobs in ODCs
associated with the same eye, but that there are significant exceptions
observed in each study. Therefore, although most of the lateral
interconnections seem to be between like compartments (e.g., blobs to
blobs; interblobs to interblobs, and interblobs within ODCs of the same
eye) a significant percentage of the axonal terminals make contact with
the CO blobs associated with the opposite eye. Such reciprocal contacts
between pairs of CO blobs could support interblob neural coherence and
provide a synchrony between ODC domains analyzing the same part of
visual space. It could be speculated further that a neural coherence is
necessary to knit together a higher order binocular perceptual map,
with the CO blobs serving as neuronal fiducial points for keeping the
monocular maps synchronized and functionally aligned. From our results,
there seems to be no differential spatial input from these sources, in
that the loss in COR appears to uniform throughout the blob. As the
afferent inputs to the blob from layers 4C and 4Cß and from the
K-cells of the LGN fail with the progressive death of the retinal
ganglion cells, the lateral input from the adjacent column driven by
the normal eye are apparently insufficient to sustains metabolic demand
within CO blobs of the glaucomatous eye. Of course, a strong counter
argument to this possibility is the absence of binocularity of neurons
within the blobs.
Experimental glaucoma produces a differential reduction in the layer 4C
input sublaminae, with a greater loss in 4Cß than in
4C.1
This result, which we reported earlier, was
consistent with what we had found in the LGN (that experimental
glaucoma had a greater effect on the metabolism of the P-cellular
pathway, than on the M-cell pathway), but incompatible with the earlier
suggestions of Glovinsky et al.,26
who presented evidence
to the contrary—that the larger parasol ganglion cells, which make up
the primate M-cell retinogeniculocortical pathway are affected first
and more severely in glaucoma in humans and in experimental glaucoma in
monkeys. The results occurring in the CO blobs reported herein suggest
a uniform reduction in COR throughout the blob, consistent with a
uniform spatial projection to the blob from the 4C and the 4Cß
sublaminae. By contrast, Edwards et al.20
have reported
recording from a small number of neurons within the center of the blob
having a higher contrast sensitivity (a characteristic of the M-cell
pathway) than those neurons near the periphery of the blob, suggesting
an anisotropy in the inputs from the M-cell and P-cell pathways. The
uniform reduction of COR throughout the blob reported here provides no
evidence for a differential spatial projection of M- and P-cell input
within the blob and is consistent with the results of Trusk et
al.14
These results further extend the description of the pathophysiology of glaucoma along the afferent chain of anatomic sites from the eye to the superficial layers of primary visual cortex.
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Acknowledgements |
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Footnotes |
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Submitted for publication May 23, 2000; revised October 10, 2000; accepted October 26, 2000.
Commercial relationships policy: N.
Corresponding author: Morris L. J. Crawford, Department of Ophthalmology and Visual Science, University of Texas Medical School at Houston, 6431 Fannin, Suite 7.024, Houston, TX 77030. morris.l.crawford{at}uth.tmc.edu
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