Histologically, retina of buffaloes showed the following layers without inwards (i) Retinal pigment epithelium (ii) Photoreceptor layer (iii) Outer limiting membrane (iv) Outer nuclear layer (v) Outer plexiform layer (vi) Inner nuclear layer (vii) Inner plexiform layer (viii) Ganglion cell layer (ix) Nerve fiber layer and (x) Inner limiting membrane (Fig 1).
Retinal pigment epithelium
In buffaloes the pigmented epithelium was cuboidal type and basally adherent to the Bruch’s membrane and apically to the photoreceptors with long projections. The cytoplasm of RPE consisted of large quantity of melanin pigment in the non tapetal portion than in the tapetal portion. The RPE cells were cuboidal in group I and gradually elongated in group III buffaloes (Fig 2a, 2b and 2c). These findings were in accordance with
Friedmann et al., (1968) in normal young human. Further, they stated that the typical RPE cells were hexagonal, mononucleated and approximately 3% of the cells of all age groups were binucleated. But in the present study the binucleated cells were not found in any age groups of buffaloes.
Melanin pigment in RPE cells was decreased from group I to III, which was supported qualitatively by immunohistological study with PAX6 antibody reaction also (Fig 3a, 3b and 3c). Drusen was not found in in the eyes of buffaloes as buffaloes obtain natural antioxidants
i.
e. Vitamin-E and Vitamin-A abundantly from their regular feed
i.
e. green grasses. This may lead to inhibit the peroxidase damage of outer segments of photoreceptors and prevents the abnormal accumulation of lipofuscin which concurrently maintain the melanin pigment balance in RPE.
Dilley and Mc Connell (1970) also mentioned that the a-tocopherol act as antioxidant and play an important role in controlling the membrane metabolism and structure of outer segment stacks of lipoprotein lamellae from peroxidation damage in animals.
The mean thickness (µm) of retinal pigment epithelium of group I, II and III was 6.06±0.18, 6.67±0.23 and 8.44±0.32 respectively (Table 1 and Fig 4). There was a significant increase in thickness of retinal pigment epithelium in group III buffaloes as compared to group I and II. It may be due to the elongation of RPE cells and accumulation of fragmented discs and metabolites of photoreceptors. Similarly,
Dellmann and Eurell (2006) stated that function of RPE includes transport of nutrients and metabolites from the capillaries of choroid to the rods and cones, phagocytosis, lysosomal degeneration and recycling of the shredded outer segments of photoreceptors.
Photoreceptors and outer nuclear layer
The photoreceptors cells were tightly packed with less intercellular spaces in group I, whereas in the group II and III intercellular spaces were (Fig 5a, 5b and 5c). The nuclei of photoreceptors from the outer nuclear layer neither displaced to outer segments of photoreceptors nor outer plexiform layers in group I buffaloes. Whereas in group II and III the nuclei of photoreceptors were extended into outer plexiform layer. Similarly
Gartner and Henkind (1981) noted displacement of photoreceptor nuclei into the outer plexiform layer in old age people.
In the present study number of rods was more compared to the cones in all age groups, but the number of cones was progressively increased and the rods were decreased from group I to group III buffaloes (Fig 5a, 5b and 5c). Similar findings were also reported by
Curcio and Drucker, (1993) and Gao and Hollyfield (1992) in human retina. These findings suggested that the rods appear to be more affected by ageing than cones both in buffalo and human. The mean thickness (µm) of photoreceptors and outer nuclear layers of retina of group I, II and III was 64.22±1.84, 49.17±1.63 and 58.72±2.1 respectively (Table 1 and Fig 4). It indicated that the mean thickness (µm) of photoreceptors and outer nuclear layers of retina of group I to III was significantly decreased due to the decreased number of rods and their nuclei with advancement of age in buffaloes. These findings suggested that the declining of sensitivity of the peripheral field of vision with ageing than that of the central field in buffaloes, which may be resulted due to the functional vulnerability of rods with ageing in animals.
Marshall et al., (1998) observed the morphological distortions in rod outer segments and their diameter was increased from 2.5 µm to 3.5 µm in all areas of retina in human.
Outer limiting membrane
The outer limiting membrane was formed by joining of adjacent photoreceptors and Muller cells processes. This membrane separated the inner segments of rods and cones from the outer nuclear layer. These findings were in accordance with the
Dellmann and Eurell (2006) in other domestic animals. The outer limiting membrane was apparent, continuous and unbroken throughout the life in all age groups of buffaloes.
Outer plexiform layer
This layer was formed by dendritic and axonic terminals of horizontal and bipolar cells along with displaced nuclei of horizontal cells in buffaloes. Similar findings were also noticed by
Dellmann and Eurell (2006) in domestic animals.
The mean thickness (µm) of outer plexiform layer of retina was 8.78±0.58, 8.28±0.33 and 10±0.51 in group I, II and III buffaloes respectively (Table 1 and Fig 4).These findings suggested that the outer plexiform layer thickness was increased with advancement of age in buffaloes. This might be due to that increased number of synaptic fibers between photoreceptors and cells of inner nuclear layer with advancement of age.
Inner nuclear layer
The inner nuclear layer consisted of four types of cells
viz., horizontal, bipolar, amacrine and Muller cells. The horizontal cells were located in the outer zone of the nuclear layer and they had round or oval nucleus and more perinuclear cytoplasm. Whereas,
Gallego and Laufer (1982) reported two types of horizontal cells
i.
e., H1 cell with a short axon and an axon less H2 cells in most of the vertebrate retinae. But in the present study such differentiation was not observed in horizontal cells of retina of buffaloes. The bipolar cells were characterized by large, round nuclei and long processes and they were placed in the center of the inner nuclear layer. The amacrine cells were predominate in the inner portion of the inner nuclear layer and their nuclei were generally characterized by deep invaginations. The Muller or radial glial cells were fibrous astrocytes characterized by a homogenous oval nuclei and a dark cytoplasm located in the outer portion of the inner nuclear layer. The density of the nuclei of these three cells in inner nuclear layer was decreased with advancement of age
i.
e. from group I to group III animals (Fig 6a, 6b and 6c). The mean thickness (µm) of inner nuclear layer was 30.83±1.48 in group I, 24.55±0.71 in group II and 22.56±0.62 in group III buffaloes (Table 1 and Fig 4). The above findings suggested that in retina the density of nuclei of different cells decreased gradually with advancement of age in inner nuclear layer. No supporting literature is available pertaining to above findings in domestic animals.
Inner plexiform layer
In buffaloes, the inner plexiform layer was a synaptic layer comprised of predominately the axons of amacrine and bipolar cells and dendrites of ganglion cells in buffaloes. These observations were coincided with the findings of
Dellmann and Eurell (2006) in domestic animals. In young animals between 1 to 2 years this layer was comprised of many displacing amacrine cells and few ganglion cells, but sparse in old buffaloes. Both connective tissue and nerve fibers were densely arranged in group I but were sparse in group II to III, this may be due to increased cystoid spaces as age advances (Fig 7a, 7b and 7c).
The mean thickness (µm) of inner plexiform layer of retina was 42.44±2.23, 32.56±0.95 and 45.39±0.81 in group I, II and III, respectively (Table 1 and Fig 4). The present observations revealed that the thickness of inner plexiform layer was increased in buffaloes in group III. Similar findings were also noticed by
Folberg (1996) and
OMalley and Allen (1967) in human retina. This degeneration was thought to be due to choroidal vascular insufficiency as opined by
OMalley and Allen (1967)) in human beings.
Ganglion cell, nerve fiber layer and inner limiting membrane
Two types of ganglion cells were reported
i.
e. a- and b-ganglion cells in the retina of buffaloes. The a-ganglion cells were large and characterized by large nuclei and less amount of cytoplasm. Whereas, the a-ganglion cells were small and consisted of small nuclei and comparatively abundant cytoplasm (Fig 7a, 7b and 7c). Similarly,
Bloom and Fawcett (1970) and
Kelly et al., (1971) also found two types of ganglion cells
i.
e. midget ganglion cells and diffuse ganglion cells.
Germain et al., (2010) identified 30 types of ganglion cells based upon their morphological differences and Boycott and Wassle, (1974) found three different types
viz., a, b and g ganglion cells in human retina on the basis of dendritic fields. The b-ganglion cells were predominant when compared with a- ganglion cells in group I, whereas with advancement of age the a- anglion cell population was relatively more in group II and III. These findings were in agreement with the findings of
Boycott and Wassle (1974) in cats.
Few melanin granules were also reported in ganglion cells. Similarly,
Dellmann and Eurell (2006) have noted melanopsin photopigment in ganglion cells of rodents and primates. The number of ganglion cells were decreased from group I to group III. Their number was approximately 16-20 cells/sq.mm in group I and 7-10 cells/sq.mm in group III animals (Table 2 and Fig 8). Similarly,
Gao and Hollyfield (1992),
Curcio and Drucker (1993) and
Grunwald et al., (1993) reported 16-20% decrease in average ganglion cell population in human from young to adults. In buffaloes the size of the both a- and the b-ganglion cells was increased from group I to III. The average size of the ganglion cell was 4.72±0.49 in group I, 9.03±0.41 in group II and 15.83±0.83 in group III (Table 2 and Fig 8). This indicated that the size of the ganglion cells was increased but their number was decreased with advancement of age.
Cavallotti et al., (2001) and
Cavallotti et al., (2004) also reported that there was an increase in size of the ganglion cells with advancement of age in rat and in human respectively.
In buffaloes the nerve fiber layer consisted of the unmyelinated axons of ganglion cells, they were surrounded by Muller cell processes and occasionally by astrocytes. The stained bodies called corpora amylacea were also present in the retina of aged buffaloes between the fibers (Fig 9). Similarly,
Avendano et al., (1980) and
Woodford and Tso (1980) also noted corpora amylacea in the pericapillary retinal nerve fiber layer, optic disc and optic nerve. Furthermore, these bodies represent the degenerated nerve cells and their organelles in ageing changes of retina of human.
Numerous blood vessels were noted in the inner plexiform and nerve fiber layers of retina in buffaloes. These blood vessels became wide and thickened due to the accumulation of fibrous tissue from group I to group III (Fig 10). It is in conformity with the findings of
Folberg (1996) in human.
Cogan (1963),
Kuwabara and Coagan (1965) and
Ramirez et al., (2001) reported arteriosclerotic changes in retinal blood vessels with ageing in human, The inner limiting membrane was loose and interrupted in group I and became thick and uninterrupted in most of the aged buffaloes (Fig 11a, 11b and 11c). Similarly,
Dellmann and Eurell (2006) also noted continuous layer of inner limiting membrane in domestic animals
In the present study the total thickness (µm) of retina in group I, II and III buffaloes were 235.5±7.25, 184.33±3.64 and 201.05±5.15 respectively (Table 1 and Fig 12). Similar findings were also reported by
Cavallotti et al., (2001) in rat and
Cavallotti et al., (2004) in human retina. The above values indicated that there was a decrease in the thickness of retina from group I to group II animals. But the total thickness of retina is slightly increased in group III.
