Distribution of αB-Crystallin in the Central Retina and Optic Nerve Head of Different Mammals and its Changes during Outer and Inner Retinal Degeneration

The small heat shock proteins alpha B-crystallin (ABC) and Hsp27 are chaperone molecules that maintain the integrity of intracellular processes during various types of stress. While Hsp27 is rather specific for the human and has only modified related proteins in other mammals (e.g. Hsp25 in rodents and birds) [1,2], ABC is a highly conserved protein that exists in nearly any species examined [3-6]. In previous work we could show that ABC is present in the retrolaminar optic nerve of numerous species including human, monkey, cow, pig, rat, and mouse [7,8]. The amount of ABC showed species differences but was homogenous within different age groups of a single species.


Introduction
The small heat shock proteins alpha B-crystallin (ABC) and Hsp27 are chaperone molecules that maintain the integrity of intracellular processes during various types of stress. While Hsp27 is rather specific for the human and has only modified related proteins in other mammals (e.g. Hsp25 in rodents and birds) [1,2], ABC is a highly conserved protein that exists in nearly any species examined [3][4][5][6]. In previous work we could show that ABC is present in the retrolaminar optic nerve of numerous species including human, monkey, cow, pig, rat, and mouse [7,8]. The amount of ABC showed species differences but was homogenous within different age groups of a single species. Under pathological conditions the expression level of ABC frequently increases. For this reason it is considered to be a useful marker in a variety of neurodegenerative diseases [9]. Concerning ocular pathologies, an increase in ABC was described in the retrolaminar optic nerve of glaucomatous monkey eyes [8]. In vitro studies confirmed the increase of ABC in human optic nerve head astrocytes exposed to elevated hydrostatic pressure [10]. In the retina, intense light exposure increased the amount of ABC in rod outer segments and retinal pigment epithelium [11]. During retinal degeneration in the rd mouse the increased ABC expression was associated with glial cell reaction [12]. Only recently, ABC secretion was described from the retinal pigmented epithelium and a neuroprotective role suggested [13].
The present study was designed to answer three major questions. A. Are there species differences in the distribution and localization of ABC in the normal retina and optic nerve head region? B. Is an increase of ABC a general phenomenon during retinal degeneration? C. Is there a difference in ABC labeling between outer (photoreceptor type) and inner (ganglion cell type) retina degeneration? 12 months of age; RCS -/-) and eight Abyssinian cats with various stages of retinal degeneration (1-8 years of age) were available. To study eyes with inner retinal degeneration, nine DBA2/NNia mice (2 to 23 months of age) and seven cynomolgus and rhesus monkeys (12-32 years of age) with unilateral laser-induced ocular hypertensive eyes were available.
Sagittal 14µm thick cryo-sections through the central retina and the optic nerve head region were performed and mounted on glass slides. Incubation with a polyclonal rabbit anti alphaB-crystallin antibody (dilution 1:400; antibody kindly provided by Hans Bloemendal, Nijmegen, The Netherlands) or a polyclonal rabbit anti glial fibrillary acidic protein (GFAP; dilution 1:500; BioGenex Laboratories, San Ramon, CA, USA) was performed over night in a moist chamber at room temperature. After rinsing in PBS, a fluorescein-conjugated goat anti rabbit Cy3 antibody (dilution 1:1000; Dianova, Hamburg, Germany) was added for 1 hour. The slides were rinsed again in PBS, mounted with Kaiser's glycerin gelatine and viewed with a fluorescence microscope (Aristoplan; Leica) at 40X to 400X magnifications.

Species differences in normal eyes with no ocular pathology
Immuno fluorescence labeling of ABC revealed clear species differences in the retina and optic nerve head: In the rat optic nerve, cell processes in the lamina cribrosa and prelaminar region labeled for ABC (Figure 1a and 1c). The estimated density of these processes was less than that of processes labeled for GFAP (Figure 1e and 1f). In the innermost layer of the papilla region towards the inner limiting membrane, consistent labeling for ABC and GFAP was seen. Only single processes, presumably of astrocytes, in the nerve fiber layer (NFL) of the retina labeled for ABC; the Muller cells were not stained ( Figure 2a). The distribution of GFAP was more intense in the NFL compared to ABC and indicated astrocytes and inner Muller cell processes (Figure 2c). Consistent labeling for ABC was found in optic nerve head sections of the cat, pig and cow. Numerous positive staining was seen in the lamina cribrosa and prelaminar region of the optic nerve ( Figure 3a  In normal monkey optic nerve specimens, the labeling distribution of ABC was comparable to that found in the cat, pig and cow ( Figure  4a and 4c).

Eyes with inherited outer retinal degeneration
In the dystrophic RCS -/-rat, labeling for ABC in the retro laminar optic nerve was similar to that observed in the normal animals. No changes occurred during retinal degeneration. At the innermost layer of the papilla region, an increase in labeling intensity was seen in the 12 months old dystrophic animals (Figure 1b

Eyes with inner retinal degeneration (glaucomatous eyes)
In rhesus monkeys, increase in intraocular pressure lead to an increase in ABC and GFAP staining mainly in the innermost layer of the papilla region towards the inner limiting membrane (Figure 4b and 4d). This increase was consistent in all glaucomatous papillae studied, while the labeling intensity of the neuronal prelaminar and lamina cribrosa region showed inter-individual differences that were not correlated to the duration or severity of glaucomatous damage. In the central retina, labeling for ABC was restricted to the astrocytes in the NFL. No increase was noted in different stages of glaucomatous damage.
In the DBA2NNia strain, the overall distribution of ABC was not changed even in old animals with late stages of glaucomatous damage. The labeling was restricted to the retroocular myelinated part of the optic nerve. No labeling appeared in the papilla region or in the retina even in 20 months old animals with severe loss of retinal ganglion cells and optic nerve fibers (data not shown). There was also no visuable increase of ABC in the retina.
A semi-quantitative analysis summarizing the above described distribution of ABC is outlined in Table 1.

Discussion
1. The results of the present study show clear differences in the distribution of ABC in the retina and optic nerve head region between the different mammalian species studied. 2. During photoreceptor degeneration, an increase in ABC was only seen in late stages when the inner layers of the retina were also affected. 3. Retinal ganglion cell degeneration led to a specific early staining of the inner prepapillary region in the monkey glaucoma model. A general problem when comparing different species and diseases is the affinity of the antibodies used. Proteins can become modified   with age, truncations can occur in degenerative conditions, and thus compromise the affinity. Unfortunately, there is no way to exclude these processes which could lead to misinterpretation. The following discussion is based on the described results and therefore reflects the actual staining performed with the specific antibody. Since the results do not contradict other statements and descriptions of ABC, they might reflect the real situation. Ad 1. Species differences were prominent between rodents and the other animals investigated in this study. Since chaperone molecules are highly related to various types of stress, a pure size-dependence of the eye (small versus large eyes) does not sufficiently interpret the findings. Two stress factors might more likely be causative: one is the absolute life span of the animals, the other one is the normal activity rhythm (nocturnal versus diurnal). A counter-example for the latter hypothesis is the fact that intense light exposure was shown to stimulate ABC expression in rod outer segments but not in the inner retina [11].
For all animals it was interesting to note, that there was no ABC staining located within the neurons, but only within glial cells (constantly astrocytes, occasionally Muller cells). This is in contrast to other crystallins and small heat shock proteins which were also described in photo receptor cells and retinal ganglion cells [25][26][27][28][29][30]. On the other hand, the close relation of ABC to glial cells is not unique in the retina but also known in other neuronal tissues [9,31]. Ad 2. Even though retinal degeneration leads to an imbalance of the local homeostasis and therefore to stress-like conditions, increase of ABC is not an early phenomenon during outer retinal degeneration. In this type of retinal degeneration, the inner layers are well preserved for a long time. Only at late stages morphological alterations are paralleled by a different expression of ABC. Heat shock protein protection seemed to appear earlier than fibrillar activation in glial cells, at least in the Abyssinian cat model. Increase of ABC was also reported in Muller cells and retinal astrocytes of the rd mouse with an accelerated type of degeneration [12].
Concerning other small heat shock proteins, a reduction of alphaAcrystallin seems to be present in the RCS rat already in early stages of the degeneration [32]. This lack of alphaA-crystallin might lead to a higher vulnerability of the photo receptors [33]. Ad 3. Increase of ABC in glaucomatous optic nerves was reported [8,10], but a precise localization has not been described so far. Interestingly, the mouse glaucoma model is not suitable for this type of investigations since ABC seems to play no major role in the mouse eye pathology. It is tempting to speculate that heat shock protein 27 might sufficiently serve the protective function in this species [34,35] and possibly in the rat, too [36].
The increase of ABC in the monkey glaucoma model showed a specific location at the inner papilla already in early stages of the disease. This unique location was not reported for other heat shock proteins [37] and might reflect a specific vulnerable region. It supports the hypothesized pathogenesis by Osborne [38] claiming the prelaminar region as the most sensitive part of the optic nerve fibers. An early and intense reaction of the local glial cells seems to take place, expressing not only ABC, but also GFAP and other factors like TGFbeta [39].
Numerous studies report an increase of ABC within the first few days following an acute insult (diabetes, retinopathy of prematurity, ischemia; reviewed in Fort and Lampi [40]) or optic nerve damage [41,42] but long term regulation is often neglected. In age-related macular degeneration ABC accumulation was described in drusen [43][44][45] and recent interest is focused on this protein in the retinal pigmented epithelium [46,47]. Increase of ABC was reported in the glaucomatous retrolaminar optic nerve [8] and trabecular meshwork [48] and in a mouse model of outer retinal degeneration [12]. In this study, several models with chronic retinal destruction were studied concerning their distribution of ABC. They show that during the process of outer retinal degeneration ABC expression is not greatly altered, suspecting no major protective mechanism of this molecule under these in vivo conditions. In contrast, the glaucoma monkey model showed an increase of ABC in the inner layer of the optic nerve head already in early stages of increased intraocular pressure which remained present in later stages. The early induction at this location points to an active role of ABC in vivo. Although effects of ABC are reported in and discussed for the retina [49], both types of retinal degeneration showed almost no changes of ABC labeling in the retina itself.
Since this study shows only mild and focused changes of ABC in a number of animal models with inner and outer retinal degeneration, future studies are required to more precisely describe the role of ABC in chronic, long-lasting situations. Chronic retinal diseases with vascular  Table 1: Semiquantitative evaluation of alphaB-crystallin distribution in the retina and optic nerve head of various animals (n= number of eyes evaluated). 0 = no staining; (+) = single or inconsistent staining; + = consistent mild staining; ++ = consistent moderate staining; +++ = consistent strong staining. e = early stages of disease, l = late stages of disease.
involvement (e.g. diabetes) might help to further understand the role of ABC, since new data points to a vascular regulating role of ABC [50] which might be more important than pure neuronal aspect. Induction of diabetes in rats showed an early increase of alpha-crystallins with a reduction at later time points [51], supporting the low levels described only recently in the human [52]. The vascular aspect, however, was not taken into account in these studies.