Immunohistochemical localization of heat shock protein 27 in the retina of pigs

The expression of heat shock protein 27 (HSP27) was examined in the retinas of pigs. Western blot analysis detected the expression of HSP27 in the retinas of 1-day-old piglets and showed that it was enhanced in the retinas of 6-month-old adult pigs. Immunohistochemically, HSP27 immunostaining was seen mainly in ganglion cell bodies in the ganglion cell layer, and in some processes of astrocytes in the innermost nerve fiber layer. In 1-day-old piglets, HSP27 was detected weakly in the inner plexiform, inner nuclear cell, outer plexiform, and rod and cone layers. The HSP27 immunoreactivity across the retinal layers was enhanced in the retinas of 6-month-old pigs compared with newborn piglets. The HSP27 immunoreactivity in the radial processes of Müller cells was particularly prominent in adult pig retinas. In summary, this finding suggests that HSP27 plays an important role in signal transduction of glial cells and neuronal cells in the retina.     

Differential expression of heat shock protein mRNAs under in vivo glutathione depletion in the mouse retina

Heat shock proteins (HSPs) are highly conserved proteins playing a protective role under deleterious conditions caused by a wide variety of pathophysiological, including environmental stresses. Glutathione (GSH) is known to play a critical role in the cellular defense against unregulated oxidative stress in mammalian cells including neurons. We previously demonstrated that GSH depletion induced cell death in the retina, but the mechanism(s) of cellular protection were not clear. Unregulated oxidative stress was induced by depletion of intracellular GSH by systematic administration of buthionine sulphoximine (BSO), an inhibitor of gamma-glutamylcysteine synthetase. After 0, 1, 4 and 7 days of BSO administration, we examined expression of both large and small HSP mRNAs (hsp90alpha, hsp90beta, hsp70, hsp60 and hsp25) in oxidative-stressed mouse retina. Of large HSPs, only hsp70 expression was significantly decreased from 1 day after BSO injection, whereas expression of other large hsps was not changed on day 1. Expression of hsp60 decreased on 4 days, whereas expression of hsp90 decreased on 7 days after BSO administration. Different from large HSPs, a small HSP, hsp25 increased its expression to a great extent from 1 day after BSO administration. Taken together, our results show that unregulated oxidative stress could induce differential expression of HSPs, which, in turn, may play distinct roles in the cellular defense. Targeting HSPs, therefore, may provide novel tools for treatment of retinal degenerative diseases such as glaucoma, retinopathy or age-related macular degeneration.     

Differential expression of beta-amyloid precursor and Bcl-2 proto-oncogene proteins in the developing dog retina

Previous studies of rat retinas have not only provided evidence that beta-amyloid precursor (APP) and B-cell lymphoma proto-oncogene (Bcl-2) proteins are colocalized in retinal Müller glial cells, but have also indicated that common mechanisms regulate their expression in these cells (Chen, S.T., Garey, L.J., Jen, L.S., 1994. Bcl-2 proto-oncogene protein immunoreactivity in normally developing and axotomised rat retinas. Neurosci. Lett. 172, 11 14; Chen, S.T., Gentleman, S.M., Garey, L.J., Jen, L.S., 1996. Distribution of beta-amyloid precursor and B-cell lymphoma proto-oncogene proteins in the rat retina after optic nerve transection or vascular lesion. J. Neuropathol. Exp. Neurol. 55, 1073-1082; Chen, S.T., Garey, L.J., Jen, L.S., 1997. Expression of beta-amyloid precursor protein immunoreactivity in the retina of the rat during normal development and after neonatal optic tract lesion. NeuroReport 8, 713-717). This investigation attempts to resolve whether or not the pattern observed in rats also applies to other higher mammalian species by examining the expression of immunoreactivity to APP and Bcl-2 in developing as well as mature dog retinas using immunocytochemical methods. Experimental results indicate that the immunoreactivity of both APP and Bcl-2 is located primarily in the inner retina, particularly in the ganglion cells and their axons in late fetal and neonatal stages. From the second postnatal week (the time of eye opening) onwards, immunoreactivity to APP, but not Bcl-2, is localized primarily in the endfeet and proximal part of the radial process of retinal Müller glial cells. Although the findings show both APP and Bcl-2 are expressed in ganglion cells and their processes suggest that the molecules have a role in the differentiation of neurons in the central nervous tissue, the lack of Bcl-2 in the Müller glial cells in dog retinas further suggests that the two molecules may have different biological roles with respect to glial function.     

Reticulon3 expression in rat optic and olfactory systems

Reticulon3 (RTN3), which belongs to a reticulon family, is first isolated from the retina, but little is known about its function. We investigated the distribution of RTN3 in rat retina and olfactory bulb by immunohistochemistry. In the retina, Müller cells highly expressed RTN3. The expression level of RTN3 in the optic nerve was high in the embryo, but low in the adult. In the olfactory system, RTN3 was highly expressed in the olfactory nerve both in developmental and adult stages. Further, RTN3 was co-localized with synaptophysin in tubulovesicular structures in the developing axon of cultured cortical neurons. These results suggest that RTN3 may play an important role in the developing axons and also in some glial cells such as Müller cells.     

外伤性视神经损伤后视神经及视网膜形态的动态观察

目的:了解外伤性视神经损伤后的病理变化、溃变特点与时相间的关系。方法:参照Allen脊髓损伤法,造成视神经眶尖段间接600gcm力冲击、挤压伤。伤后对视神经和视网膜行形态学动态观察。结果:①伤后48h,视神经轻度肿胀和空泡反应;1周时损伤处视神经出现溃变,神经胶质细胞增生,视网膜神经节细胞(retinalganglioncells,RGCs)形态改变不明显;2周时神经纤维轴束间空泡样改变,局灶性坏死,RGCs核固缩和细胞数量减少。术后3月,视神经损伤部位直径缩小,形成胶质疤痕,RGCs数量明显减少,核固缩细胞增多。②RGCs数量于术后48h、1周、2周、1月和3月分别比正常对照组低3.35%、13.23%、19.74%、23.20%、29.28%。③视网膜细胞在48h内出现凋亡。结论:本实验模型可造成明确的视神经和视网膜损伤,神经元的损伤程度从节细胞、中间神经元、感光细胞的次序依次递减。视网膜和视神经损伤的严重程度与时间呈相关性。RGCs数量在48h至1周时下降速率最快。     

Highly sialylated N-CAM is expressed in adult mouse optic nerve and retina

Summary The localization of the neural cell adhesion molecule (N-CAM) and its highly sialylated form, which is prevalent in young tissues and has therefore been called embryonic neural cell adhesion molecule, was studied in the developing and adult mouse optic nerve and retina immunohistologically and immunochemically. At embryonic and early postnatal ages, neuroblasts and young postmitotic neurons, Müller cells and astrocytes in the retina, and retinal ganglion cell axons and all glial cells in the optic nerve express highly sialylated neural cell adhesion molecule. Beginning with the third postnatal week, highly sialylated neural cell adhesion molecule disappears from retinal ganglion cell axons in the optic nerve and from neuronal cell bodies and processes in the retina. In addition, it is not detectable on oligodendrocytes in 3-week-old animals. However, highly sialylated neural cell adhesion molecule continues to be expressed in the adult optic nerve and retina by astrocytes and Müller cells. On these cells it is only absent from cell membranes contacting basal lamina. Weakly sialylated neural cell adhesion molecule, in contrast, is expressed by all cell types of retina and optic nerve during development and in the adult. The loss of highly sialylated neural cell adhesion molecule from neurons and oligodendrocytes must therefore be considered as a cell type-specific conversion of the so-called embryonic to the adult form of neural cell adhesion molecule and does not simply reflect the disappearance of neural cell adhesion molecule from these cells. Weakly sialylated neural cell adhesion molecule, however, is absent from outer segments of photoreceptor cells and, as is the case for the highly sialylated form, from glial cell surfaces contacting basal lamina. Thus, the expression of highly sialylated neural cell adhesion molecule by pre- and postmitotic neurons and by oligodendrocytes is restricted mainly to the period of histogenetic events in retina and optic nerve, i.e. cell division, cell migration, dendritic and axonal growth and synaptogenesis. In addition to the observation that this form of neural cell adhesion molecule is less adhesive than the weakly sialylated, adult form, it is likely that highly sialylated neural cell adhesion molecule plays an important role during dynamic morphogenetic events. Furthermore, the expression of highly sialylated neural cell adhesion molecule by astrocytes and Müller cells in adult optic nerves and retinae suggests some histogenetically plastic functions for these cells in the adult mouse visual system.     

Cilostazol promotes survival of axotomized retinal ganglion cells in adult rats

Cilostazol (CLZ), a selective inhibitor of cyclic nucleotide phosphodiesterase 3, has been shown to reduce neuronal cell death after a transient cerebral infarction. The mechanism for this reduction was suggested to be an elevation of intracellular cAMP or an inhibition of tumor necrosis factor alpha. Optic nerve injury leads to retinal ganglion cell (RGC) death possibly from a deprivation of neurotrophic factors and/or the down-regulation of intracellular cAMP. The purpose of this study was to determine if CLZ can rescue RGCs after optic nerve transection by inhibiting cyclic nucleotide phosphodiesterase 3. To examine this, the mean densities of surviving RGCs after optic nerve transection were determined in retinas that received an intravitreal injection of CLZ and in retinas that received vehicle. Our results showed that the density of surviving RGCs in the retina with intravitreal CLZ were significantly higher than that with vehicle injection on day 7. The CLZ was effective in promoting the survival at more than 0.05% concentration. The neuroprotective effects induced by 0.05% CLZ could be observed even 14 days after optic nerve transection. Furthermore, combined application of protein kinase A (PKA) inhibitor, KT5720 (10 microM) and 0.05% CLZ significantly decreased the density of surviving RGCs compared to that with only 0.05% CLZ. Based on these data, we concluded that CLZ enhances the survival of axotomized RGC in vivo, possibly depending on the activation of PKA pathway.     

Expression of the 40 kDa catecholamine regulated protein in the normal and injured rat retina

Catecholamine regulated protein 40 (CRP40) has been shown to be expressed in the central nervous system (CNS) of several mammalian species where it may function in a similar manner to members of the heat shock protein (HSP) family. Immunohistochemical and immunoblotting techniques were utilized to investigate whether CRP40 is expressed in normal rat retinas. In addition, changes in CRP40 expression were studied following optic nerve transection. The immunohistochemical results showed that CRP40 is expressed in the normal rat retina. The protein was found to be highly expressed in the ganglion cell layer (GCL), the inner nuclear layer (INL) and the outer plexiform layer (OPL). In addition, a low level of CRP40 was found in the inner plexiform layer (IPL), and in the inner segment layer (ISL). No expression was found in the outer nuclear layer (ONL) of normal rat retina. The immunoblotting results show that CRP40 expression decreased in a time-dependent fashion after the optic nerve transection. This decrease indicates that the expression of CRP40 is dependent on the neuron's normal physiological state and that it plays an important function in physiological and pathological conditions in the retina.     

The serine racemase mRNA is expressed in both neurons and glial cells of the rat retina

D-Serine, an endogenous and obligatory coagonist for the glycine site of the N-methyl-D-aspartate receptor in mammals, is synthesized from L-serine by serine racemase. Serine racemase and D-serine have long been believed to occur predominantly in astrocytes, according to immunohistochemical studies. Recent studies have demonstrated, however, that both the mRNA and protein levels of serine racemase are considerably higher in neurons than in astrocytes in primary cultures of the rat brain and that the mRNA level of serine racemase predominates in neurons of the adult rat brain. Here we report the application of in situ hybridization based on tyramide signal amplification for the detection of serine racemase mRNA in sections of the adult rat retina and optic nerve head. The localization of serine racemase mRNA could be demonstrated in ganglion cells, amacrine cells, bipolar cells, horizontal cells, and Müller cells of the retina as well as in the astrocytes of the optic nerve head and the lamina cribrosa. This is the first study to demonstrate the exact localization of serine racemase mRNA at the cellular or tissue level in the retina and the optic nerve head. These results suggest that both the neuron- and glia-derived D-serine could modulate neurotransmission via the glycine site of the N-methyl-D-aspartate receptors in the retina.     

The expression of heat shock protein 27 in retinal ganglion and glial cells in a rat glaucoma model

The heat shock protein 27 kDa (HSP27) is a member of proteins that are highly inducible under various forms of cellular stress. This study describes constitutive HSP27 expression in rat retina and stress-associated expression of HSP27 in an experimental rat glaucoma model. Glaucoma was induced unilaterally using laser photocoagulation of the episcleral and limbal veins. Three and seven days after the elevation of intraocular pressure (IOP), groups of rats were killed. The second laser treatment was performed for those rats killed 14 and 21 days after the first laser treatment. The RGCs were labeled with a retrograde tracer 7 days before kill. The expression of HSP27 was analyzed by Western blotting in retinas of rats killed on day 14 after the first laser treatment. Retinal astrocytes, Müller cells and HSP27-positive cells were visualized using immunohistochemical methods both from retinal whole-mounts and paraffin sections. The total number of retrogradely labeled RGCs decreased by 23.2% after 7 days, 28% after 14 days, and 29.3% after 21 days of elevated IOP when compared with controls. A significant decrease of glial fibrillary acidic protein (GFAP)-immunoreactive retinal astrocytes in laser-treated eyes was observed compared with the controls (accounted for 44.9%, 38.2% and 35% of the control values in the 7-day, 14-day and 21-day groups, respectively). The expression of HSP27 in RGCs and retinal astrocytes was also increased in laser-treated eyes when compared with controls in all groups. However, glycinergic and cholinergic cells in the inner nuclear layer and the highest number of RGCs and astrocytes that expressed HSP27 were found in the 14-day group of rats. The constitutive expression of HSP27 was observed only in retinal astrocytes and Müller cells. This study suggests that constitutive HSP27 expression is a cell-type specific phenomenon in the rat retina. However, at the same time, HSP27 might be considered as a marker for neuronal injury in the rat glaucoma model.     

Expression of heat shock proteins in osteosarcoma and its relationship to prognosis

The prognosis of osteosarcoma has been improved by chemotherapy. Heat shock proteins (HSPs) assist in folding proteins at posttranslation and degeneration under stress. We investigated the effect of HSPs on survival in osteosarcoma. Conventional osteosarcomas of the extremities from 70 patients aged 30 years or younger were used. Preoperational chemotherapy was performed in all cases. Tissues at surgery and biopsy were immunohistochemically stained with anti-HSP27, HSP47, HSP60, HSP70, HSP90alpha, HSP90beta, and p53 antibodies. We classified the cases in which more than 10% of tumor cells were positive into the overexpressing group. Overall survival was compared between the groups either overexpressing HSPs or not using Wilcoxon's test and Cox's proportional hazard model. The overexpression rate at biopsy was 22% (HSP27), 88% (HSP47), 66% (HSP60), 48% (HSP70(, 47% (HSP90alpha), 31% (HSP90beta), and 17% (p53), respectively. The rate at surgery was 33% (HSP27), 94% (HSP47), 60% (HSP60), 49% (HSP70), 28% (HSP90alpha), 40% (HSP90beta), and 17% (p53), respectively. HSP27 and p53 overexpression at biopsy had a negative prognostic value. HSP27 showed the strongest negative prognostic value in osteosarcoma. It is therefore important to investigate further its function in cellular regulation and drug resistance.     

Upregulation of retinal transglutaminase during the axonal elongation stage of goldfish optic nerve regeneration

Fish CNS neurons can repair their axons following nerve injury, whereas mammalian CNS neurons cannot regenerate, and become apoptotic within 1-2 weeks after the nerve lesion. One explanation for these differences is that one, or several molecules are upregulated in fish CNS neurons during nerve regeneration, and this same molecule is downregulated in mammalian CNS neurons before the development of apoptosis caused by nerve injury. A molecule satisfying these criteria might successfully rescue and repair the mammalian CNS neurons. In this study, we looked for such a candidate molecule from goldfish retinas. Transglutaminase derived from goldfish retina (TG(R)) was characterized as a regenerating molecule after optic nerve injury. A full-length cDNA for TG(R) was isolated from the goldfish retinal cDNA library prepared from axotomized retinas. Levels of TG(R) mRNA and protein increased only in the retinal ganglion cells (RGCs) between 10 and 40 days after optic nerve transection. Recombinant TG(R) protein enhanced neurite outgrowth from adult fish RGCs in culture. Specific interference RNA and antibodies for TG(R) inhibited neurite outgrowth both in vitro and in vivo. In contrast, the level of TG(R) protein decreased in rat RGCs within 1-3 days after nerve injury. Furthermore, the addition of recombinant TG(R) to retinal cultures induced striking neurite outgrowth from adult rat RGCs. These molecular and cellular data strongly suggest that TG(R) promotes axonal elongation at the surface of injured RGCs after optic nerve injury.     

Ischemia-reperfusion alters the immunolocalization of glial aquaporins in rat retina

Glial cells control the retinal osmohomeostasis, in part via mediation of water fluxes through aquaporin (AQP) water channels. By using immunohistochemical staining, we investigated whether ischemia-reperfusion of the rat retina causes alterations in the distribution of AQP1 and AQP4 proteins. Transient ischemia was induced in retinas of Long–Evans rats by elevation of the intraocular pressure for 60 min. In control retinas, immunoreactive AQP1 was expressed in the outer retina and by distinct amacrine cells, and AQP4 was expressed by glial cells (Müller cells and astrocytes) predominantly in the inner retina. After ischemia, retinal glial cells in the nerve fiber/ganglion cell layers strongly expressed AQP1. The perivascular staining around the superficial vessels altered from AQP4 in control retinas to AQP1 in postischemic retinas. The data suggest that the glial cell-mediated water transport in the retina is altered after ischemia especially at the superficial vessel plexus.     

Nestin expression in Müller glial cells in postnatal rat retina and its upregulation following optic nerve transection

This study examined the nestin immunoexpression and its specific cellular localization in the developing retina of rats and investigated its putative changes in an altered environment. At postnatal day 0, nestin immunoexpression was detected in radially oriented cells considered to be neural progenitors that were glutamine synthetase (GS) negative. With age, it was localized in differentiating and differentiated GS positive Müller glial cells. Nestin expression was down-regulated as maturation proceeded, so that by 12 weeks, it was almost completely diminished as confirmed also by real time-polymerase chain reaction analysis. Nestin expression along with that of glial fibrillary acidic protein (GFAP) was induced and upregulated in mature Müller glial cells following optic nerve transection. It is suggested that both nestin and GFAP may be useful biomarkers in retinal injuries. In view of their cytoskeletal nature, the marked expression of nestin and GFAP may provide a structural support for the framework of retina which would be disrupted as a result of loss of neurons in optic nerve lesion. It may also be neuronal protective taking into consideration the close spatial and functional links between Müller glial cells and the axotomized ganglion cells.     

Thy-1 antigen: a ganglion cell specific marker in rodent retina

A monoclonal antibody, 2G12 , has been produced against a rat cerebral cortex glycoprotein fraction. It interacts with Thy-1 based on both the tissue distribution of its reactivity and the blocking of its binding by pretreatment with a rabbit anti-Thy-1 serum. In rat retina this antibody labels only cell bodies in the ganglion cell layer, optic nerve fibres and the inner plexiform layer. That the cell-body labelling was confined to ganglion cells was confirmed by double-labelling experiments. Ganglion cells were distinguished by retrograde transport of fluorescent markers injected into the superior colliculi. The retinas were dissociated into single cells and the cell suspension was labelled with 2G12 . There was almost complete coincidence of the two labels. A monoclonal antibody against mouse Thy-1.2 gave an essentially identical pattern of labelling. In both rats and mice Thy-1 was also found on the vitreal surface of the inner limiting membrane in a pattern reminiscent of that formed by the Müller cell endfeet, although these cells do not express Thy-1.     

Evolution of apparent diffusion coefficient and transverse relaxation time (T2) in the subchronic stage of global cerebral oligemia in different rat models

Using magnetic resonance imaging techniques, we examined the time course of apparent diffusion coefficient (ADC), T2, and T2* relaxation times in 1-year-old rats after different forms of cerebral oligemia had been induced by (1) transient systemic hypotension, (2) permanent bilateral carotid artery occlusion (BCCAO), and (3) combined hypotension and BCCAO over a time period of 14 days after the oligemic event. These groups were compared with a group of sham-operated adult rats (controls, 4) to rule out a drift of the parameters over time. The animals were examined in a 2.35 T scanner. ADC, T2, and T2* were measured in both hemispheres of rat parietotemporal cerebral cortex, thalamic nuclei, and hippocampus 1 day before as well as on days 1, 3, 7, and 14 after sham operation and in different models of oligemia, respectively. Hypotension alone had no significant effect on MRI parameters in rat brain. After BCCAO, an increase in T2* was observed. If a permanent BCCAO was combined with transient hypotension, however, 84% of 1-year-old animals died within 14 days after surgery. In the surviving animals, significant changes in ADC, T2, and T2* were observed in the hippocampus and parietotemporal cerebral cortex. ADC showed a decrease on day 1 after oligemia, and an increase on days 3, 7, and 14. The T2* and T2 values were markedly increased on days 7 and 14 after surgery. In conclusion, only severe oligemia combining BCCAO and hypotension induces significant changes in tissue integrity (as shown by ADC) and in blood oxygenation levels in the subchronic period, whereas no significant changes were detected if permanent BCCAO or transient hypotension was applied separately.     

Somatostatin-like immunoreactive material in associational ganglion cells of human retina

The retinal ganglion cell is classically viewed as the output cell of the retina, sending a single axon via the optic nerve to synapse in visual relay nuclei of the brain. However, some ganglion cells, termed associational ganglion cells, have axons which do not leave the retina and presumably serve intraretinal communication. Using high-affinity and specific monoclonal antibodies to somatostatin-14 and the avidin-biotin-peroxidase immunohistochemical procedure, somatostatin-immunoreactive associational ganglion cells are specifically stained in human retinas obtained at necropsy. These cells are more numerous in the inferior than the superior retina; they have dendrites which ramify in the inner plexiform layer; and they have sparsely branching axons, many of which can be traced over 1 cm. These axons do not enter the optic nerve. They follow remarkably straight courses at the border of the inner plexiform layer and ganglion cell layer and thereby form a gridwork of fibers covering the entire retinal area. These observations verify the existence of associational ganglion cells in the human and establish somatostatin as a neurotransmitter or neuromodulator candidate for these neurons. The morphology of these cells suggests that they are involved in long-distance interactions within the retina.     

Induced and constitutive heat shock protein expression in the olfactory system--a review, new findings, and some perspectives

Heat shock, or stress, proteins (HSPs) are cellular proteins induced in response to conditions that cause protein denaturation, and their induction is essential for survival of such conditions. In the olfactory system we have found intense HSP expression occurs during normal processing of environmental odorants/inhalants as well as following hyperthermia and drug exposure. The HSPs involved include ubiquitin, HSP70, HSC70, and HSP25. Responses are both cell type- and stress-specific, occurring primarily in olfactory supporting cells and to some extent in Bowman’s gland acinar cells. Responses to these stresses are not seen in olfactory sensory neurons. This article reviews those studies and the significance of their findings. It also discusses a distinct subpopulation of rat olfactory sensory neurons (OSNs), the 2A4(+)OSNs, found to be constitutively reactive with HSP70, the predominantly stress-inducible isoform of the 70 kD HSP family. Their high HSP70 expression appears to confer on the 2A4(+)OSNs an enhanced ability to survive damage-induced OSN turnover. New findings are also presented on HSP25-specific changes following olfactory bulbectomy. All data are discussed in the context of the overall olfactory and bioprotective functions of the olfactory mucosa.