Multi-Scale Investigation of Human Renal Tissue in Three Dimensions.

Histopathology as a diagnostic mainstay for tissue evaluation is strictly a 2D technology. Combining and supplementing this technology with 3D imaging has been proposed as one future avenue towards refining comprehensive tissue analysis. To this end, we have developed a laboratory-based X-ray method allowing for the investigation of tissue samples in three dimensions with isotropic volume information. To assess the potential of our method for micro-morphology evaluation, we selected several kidney regions from three patients with cystic kidney disease, obstructive nephropathy and diabetic glomerulopathy. Tissue specimens were processed using our in-house-developed X-ray eosin stain and investigated with a commercial microCT and our in-house-built NanoCT. The microCT system provided overview scans with voxel sizes of [Formula: see text] and the NanoCT was employed for higher resolutions including voxel sizes from [Formula: see text] to 210 nm. We present a methodology allowing for a precise micro-morphologic investigation in three dimensions which is compatible with conventional histology. Advantages of our methodology are its versatility with respect to multi-scale investigations, being laboratory-based, allowing for non-destructive imaging and providing isotropic volume information. We believe, that after future developmental work this method might contribute to advanced multi-modal tissue diagnostics.

Revealing the Microscopic Structure of Human Renal Cell Carcinoma in Three Dimensions.

For fully characterizing renal cell carcinoma (RCC), information about the 3D tissue microstructure is essential. Histopathology, which represents the current diagnostic gold standard, is destructive and only provides 2D information. 3D X-ray histology endeavors to overcome these limitations by generating 3D data. In a laboratory environment, most techniques struggle with limited resolution and the weak X-ray attenuation contrast of soft tissue. We recently developed a laboratory-based method combining nanoscopic X-ray CT with a cytoplasm-specific X-ray stain. Here, we present the application of this method to human RCC biopsies. The NanoCT slices enable pathological characterization of crucial structures by reproducing tissue morphology with a similar detail level as corresponding histological light microscopy images. Beyond that, our data offer deeper insights into the 3D configuration of the tumor. By demonstrating the compatibility of the X-ray stain with standard pathological stains, we highlight the feasibility of integrating staining based NanoCT into the pathological routine.

Antiglaucoma EP2 Agonists: A Long Road That Led Somewhere.

For >2 decades, EP2 agonists have been the subject of antiglaucoma research and development by scientists in industry and academia around the world. The road has led to the recent approval of the first drug of this class. This article reviews the development of EP2 agonists from conception to clinical approval, discussing pharmacology, structure, biodistribution, therapeutics, and drug delivery. An extensive list of source references is provided for the reader's benefit.

Activation of the PI3K/AKT pathway correlates with prognosis in stage II colon cancer.

BACKGROUND: Patients with UICC/AJCC stage II colon cancer have a high 5-year overall survival rate after surgery. Nevertheless, a significant subgroup of patients develops tumour recurrence. Currently, there are no clinically established biomarkers available to identify this patient group. We applied reverse-phase protein arrays (RPPA) for phosphatidylinositide-3-kinase pathway activation mapping to stratify patients according to their risk of tumour recurrence after surgery. METHODS: Full-length proteins were extracted from formalin-fixed, paraffin-embedded tissue samples of 118 patients who underwent curative resection. RPPA technology was used to analyse expression and/or phosphorylation levels of six major factors of the phosphatidylinositide-3-kinase pathway. Oncogenic mutations of KRAS and BRAF, and DNA microsatellite status, currently discussed as prognostic markers, were analysed in parallel. RESULTS: Expression of phospho-AKT (HR=3.52; P=0.032), S6RP (HR=6.3; P=0.044), and phospho-4E-BP1 (HR=4.12; P=0.011) were prognostic factors for disease-free survival. None of the molecular genetic alterations were significantly associated with prognosis. CONCLUSIONS: Our data indicate that activation of the PI3K/AKT pathway evidenced on the protein level might be a valuable prognostic marker to stratify patients for their risk of tumour recurrence. Beside adjuvant chemotherapy targeting of upregulated PI3K/AKT signalling may be an attractive strategy for treatment of high-risk patients.

A specific expression profile of heat-shock proteins and glucose-regulated proteins is associated with response to neoadjuvant chemotherapy in oesophageal adenocarcinomas.

BACKGROUND: Oesophageal adenocarcinomas often show resistances to chemotherapy (CTX), therefore, it would be of high interest to better understand the mechanisms of resistance. We examined the expression of heat-shock proteins (HSPs) and glucose-regulated proteins (GRPs) in pretherapeutic biopsies of oesophageal adenocarcinomas to assess their potential role in CTX response. METHODS: Ninety biopsies of locally advanced adenocarcinomas before platin/5-fluorouracil (FU)-based CTX were investigated by reverse phase protein arrays (RPPAs), immunohistochemistry (IHC) and quantitative RT-PCR. RESULTS: CTX response strongly correlated with survival (P=0.001). Two groups of tumours with specific protein expression patterns were identified by RPPA: Group A was characterised by low expression of HSP90, HSP27 and p-HSP27((Ser15, Ser78, Ser82)) and high expression of GRP78, GRP94, HSP70 and HSP60; Group B exhibited the inverse pattern. Tumours of Group A were more likely to respond to CTX, resulting in histopathological tumour regression (P=0.041) and post-therapeutic down-categorisation from cT3 to ypT0-T2 (P=0.040). High HSP60 protein (IHC) and mRNA expression were also associated with tumour down-categorisation (P=0.016 and P=0.004). CONCLUSION: Our findings may enhance the understanding of CTX response mechanisms, might be helpful to predict CTX response and might have translational relevance as they highlight the role of potentially targetable cellular stress proteins in the context of CTX response.

Characterization of an in vivo model for the study of eyelash biology and trichomegaly: mouse eyelash morphology, development, growth cycle, and anagen prolongation by bimatoprost.

BACKGROUND: Hypertrichosis or alopecia of the eyelashes is associated with various diseases or may be drug induced. Although neither increase nor loss of eyelashes is life threatening, eyelash disorders can be psychologically disturbing. However, as control of eyelash growth and the underlying mechanisms of eyelash hypo- or hypertrichosis are largely obscure, available therapy is limited. OBJECTIVES: To improve this situation, we sought to establish a pragmatic, well-defined mouse model for the study and pharmacological investigation of eyelash follicle biology. METHODS: We took a morphometric approach to establish an eyelash model using female C57BL/6J mice by comparing with pelage hairs and highlighting the differences. We next applied a hypertrichosis-triggering agent and investigated its effect using the model. RESULTS: In eyelashes, a synchronized growth cycle was observed after morphogenesis but was completed earlier than pelage hairs. Exogen was strictly regulated and occurred in every cycle in the eyelash. Otherwise, general morphological features of mouse eyelashes (shafts, follicles, morphogenesis and growth cycle) were comparable with those of pelage hairs. The eyelash growth-stimulatory agent in humans, bimatoprost, significantly extended the duration of anagen, resulting in more and longer eyelashes, but there was no evidence of follicle neogenesis. CONCLUSIONS: This study shows that mouse eyelashes offer an excellent in vivo model for the quantitative and qualitative analysis of eyelash morphology, development, growth cycle, exogen and pharmacological modulation. This model will help to elucidate the unknown molecular controls of eyelash growth, and to develop novel drugs to treat eyelash disorders.

Transient postoperative vascular endothelial growth factor (VEGF)-neutralisation improves graft survival in corneas with partly regressed inflammatory neovascularisation.

BACKGROUND: High-risk keratoplasties are usually performed after an uninflamed and quiescent interval in corneas with partly regressed blood and lymphatic vessels. We analysed whether the inhibition of post-keratoplasty revascularisation in mice with partly regressed corneal vessels ("intermediate-risk") improves graft survival. METHODS: Three interrupted stromal sutures (11-0) in corneas of Balb/c mice (6-8 weeks old) were placed for 6 weeks. Six months after suture removal, penetrating keratoplasty was performed with C57BL/6 donors. The treatment group received a vascular endothelial growth factor-A specific cytokine trap (VEGF Trap) intraperitoneally at days 0, 4, 7 and 14 after keratoplasty (25 mg/kg per mouse; controls received equal amounts of Fc protein). Pathological haemangiogenesis and lymphangiogenesis prior to as well as 3 days or 8 weeks after keratoplasty and graft survival were analysed. RESULTS: Three days after keratoplasty corneal revascularisation was sufficiently reduced by VEGF Trap (haem-vascularised areas 42.7% reduction; lymph-vascularised areas 54.7% reduction). Survival proportions 8 weeks after keratoplasty were 36% in the treatment group compared with 9% in the control group (n = 11; p<0.05). At that time no differences in haemangiogenesis or lymphangiogenesis were observed between the two groups. CONCLUSION: Early transient postoperative induction of haemangiogenesis and lymphangiogenesis and reformation of regressed corneal blood and lymphatic vessels are important for transplant rejections after "intermediate-risk" corneal transplantation.

[Primary open angle glaucoma. Morphological bases for the understanding of the pathogenesis and effects of antiglaucomatic substances]. / Primäres Offenwinkelglaukom. Morphologische Grundlagen zum Verständnis der Pathogenese und der Wirkung antiglaukomatöser Substanzen.

The pathogenesis of glaucomatic illnesses is poorly understood. An increase in ocular pressure can be caused by an increase in the secretion of aqueous humour or a reduction in its outflow. In the elderly, outflow is reduced while at the same time less aqueous humour is produced. This balance is easily disturbed, so that age represents a risk factor for glaucoma in addition to increased ocular pressure. Therapeutic possibilities involve, on the one hand, reducing the secretion of aqueous humour, for example using, beta blockers, carbonic anhydrase inhibitors and clonidine. On the other hand, aqueous humour outflow can also be influenced by drugs. Conventional outflow is increased by the administration of miotics. The uveoscleral outflow can be increased by prostaglandin derivates. Drugs which only influence trabecular outflow are not yet available. Future therapeutic possibilities involve new aspects of the pathophysiology, e.c. the use of growth factors, free radical scavenging enzymes and choroidal blood flow.

Glutathione peroxidase-1 deficiency leads to increased nuclear light scattering, membrane damage, and cataract formation in gene-knockout mice.

PURPOSE: Previous in vitro studies with transgenic and gene-knockout mice have shown that lenses with elevated levels of glutathione peroxidase (GPX)-1 activity are able to resist the cytotoxic effect of H(2)O(2), compared with normal lenses and lenses from GPX-1-deficient animals. The purpose of this study was to investigate the functional role of this enzyme in antioxidant mechanisms of lens in vivo by comparing lens changes of gene-knockout mice with age-matched control animals. METHODS: In vivo lens changes were monitored by slit lamp biomicroscopy, and enucleated lenses were examined under a stereomicroscope in gene-knockout animals and age-matched control animals ranging in age from 3 weeks to 18 months. Transmission (TEM) and confocal microscopy were performed on different regions of lenses after the mice were killed at various times. RESULTS: Slit lamp images showed an increase in nuclear light scattering (NLS) in gene-knockout mice compared with control animals. TEM revealed changes in the nucleus as early as 3 weeks of age by the appearance of waviness of fiber membranes. With increasing age, there was greater distortion of fiber membranes and distension of interfiber space at the apex of fiber cells compared with control mice. The changes in nuclear fiber membranes were even more dramatic, as observed by confocal microscopy, which was performed on thicker sections. In contrast to the changes in the lens nucleus, the morphology of the epithelium and superficial cortex remained unchanged in knockout animals during the same experimental period, consistent with slit lamp observations. Stereomicroscopy of ex vivo lenses demonstrated a significant increase in opacification in gene-knockout mice relative to control animals of the same age. This effect became evident in mice aged 5 to 9.9 months and persisted thereafter in older animals, resulting in mature cataracts after 15 months. CONCLUSIONS: The results demonstrate the critical role of GPX-1 in antioxidant defense mechanisms of the lens nucleus. The increased NLS appears to be associated with damage to fiber membranes in the nucleus, which is particularly susceptible to oxidative challenge because of the deficiency of GPX-1. It is suggested that the lens membrane changes in the knockout animals may be due to the formation of lipid peroxides, which serve as substrates for GPX-1. Cataract development in gene-knockout mice appeared to progress from focal opacities, apparent at an earlier age, to lamellar cataracts between 6 and 10 months, and finally to complete opacification in animals older than 15 months. This is the first reported phenotype in GPX-1-knockout mice.

Transforming growth factor beta 2 levels in the aqueous humor in different types of glaucoma and the relation to filtering bleb development.

PURPOSE: To investigate the transforming growth factor beta 2 (TGF-beta 2) levels and total protein levels in the aqueous humor of eyes with different types of glaucoma [primary open-angle glaucoma (POAG), pseudoexfoliation glaucoma (PSX), juvenile glaucoma (JG)], and the relation to filtering bleb development after trabeculectomy. METHODS: Aqueous humor was collected at the beginning of surgery from 52 eyes with glaucoma (29 POAG eyes, 17 PSX eyes, 6 JG eyes) and from 29 control eyes that underwent cataract operation. TGF-beta 2 levels (intrinsically activated and total TGF-beta 2) using ELISA methods as well as total protein concentrations of the aqueous humor were determined. All preoperative clinical data of the glaucoma eyes (age, gender, IOP, previous treatment, type of surgery) were compared with the TGF-beta 2 levels. In 40 of these eyes, the postoperative follow-up (filtering bleb development, need for intervention, IOP) was correlated to the preoperatively determined TGF-beta 2 levels. RESULTS: TGF-beta 2 levels were increased in nearly half of the eyes with POAG and in most of the eyes with JG, but in eyes with PSX, TGF-beta 2 levels were within the normal range. No correlation between TGF-beta 2 levels and age, gender, IOP, previous treatment, or type of surgery, or between TGF-beta 2 levels and protein levels in aqueous humor, was found. Correlation between bleb formation and TGF-beta 2 levels revealed that all but two of the POAG eyes with good clinical outcome (type 1 bleb) had normal levels of activated TGF-beta 2. Of the 13 eyes that needed postoperative intervention (type 2 and type 3 bleb), 8 had high and 5 had normal TGF-beta 2 levels. CONCLUSIONS: PSX eyes differ from POAG and JG eyes not only by their clinical or biomicroscopic appearance, but also by their normal TGF-beta 2 levels in aqueous humor. The fact that most of the POAG eyes with favorable bleb development had normal TGF-beta 2 levels indicated that there might be some relationship between bleb formation and TGF-beta 2 levels. On the other hand, the fact that eyes with less favorable bleb development had both low and high TGF-beta 2 levels indicated that other factors are also involved in the scarring of the filtration bleb.

Retinal morphology and ERG response in the DBA/2NNia mouse model of angle-closure glaucoma.

PURPOSE: To document the time course of retinal dysfunction by scotopic electroretinography (ERG) and by quantitative morphology in eyes of the DBA/2NNia substrain of mouse (DBA) with inherited angle-closure glaucoma. METHODS: DBA and control C57BL/6J (C57) mice were studied by ERG recordings from 5 to 15 months of age, and by morphology from 1 to 14 months of age. Scotopic ERGs were simultaneously recorded from both eyes of dark-adapted anesthetized mice. Changes in the central neuronal retina were evaluated by quantitative morphometry performed on serial semithin sections of Epon-embedded eyes. RESULTS: When compared with normal C57 mice, DBA mice showed significant reductions of the a-wave and b-wave amplitudes by 7 to 8 months, and the decline continued as the animals aged. The b-wave implicit time in DBA mice showed a gradual prolongation beginning at 8 months of age, when compared with C57 mice. Logistic regression analyses revealed significant correlations in a- and b-wave amplitude reductions between ipsilateral and contralateral eyes of DBA mice at ages when ERG parameters were greatly altered. Morphologically, thinning of the whole retina was already evident in DBA mice at 4 months of age, but loss of ganglion cells and thinning of the outer plexiform layer were first seen in 7- to 8-month-old animals. These changes progressed to the end of the 13-month period studied. CONCLUSIONS: Progressive thinning of the outer retinal layers in DBA mice was found to correlate with decreases in ERG a- and b-wave amplitudes, both occurring from the age of 7 to 8 months onward. Similarities with the findings in human late-stage glaucomatous retinopathy indicate the relevance of this animal model in further glaucoma research.

Beta-adrenergic blocker therapy and the trabecular meshwork.

PURPOSE: To determine whether beta-adrenergic blocker (beta-blocker) therapy for glaucoma causes changes in the trabecular meshwork due to underperfusion. METHODS: Thirty-five eyes from 19 donors with primary open-angle glaucoma (POAG) were divided into three groups: eyes receiving beta-blocker therapy along with standard medications, eyes receiving standard medications but no beta-blockers, and eyes with elevated intraocular pressure but receiving no therapy. Transmission electron microscopy was used to assess the extracellular material of the cribriform region, the structure of the trabecular lamellae, and pigmentation of the trabecular cells. Six eyes from four normal donors were used as controls. RESULTS: No specific changes in the trabecular meshwork were found in eyes receiving beta-blocker therapy. The amount and composition of the extracellular matrix of the cribriform region and the morphology of the lamellae were similar among the three groups of eyes with POAG. Pigmentation of trabecular cells appeared to be a marker for aqueous flow, as significantly more cells contained pigment in regions of the meshwork with thin or normal lamellae than in regions with thickened and fused lamellae. These regions were variable around the circumference of the eye, and were similar between eyes with and without beta-Blocker therapy. CONCLUSION: beta-Blocker therapy could not be proven to cause underperfusion changes in the trabecular meshwork or other discernible effects. Preferential pathways for aqueous flow probably exist within regions of the trabecular meshwork, as evidenced by lamellar appearance and pigmentation of the adjacent trabecular cells.

Evaluation of the rhodopsin knockout mouse as a model of pure cone function.

PURPOSE: To determine a time window in the rhodopsin knockout (Rho(-/-)) mouse during which retinal function is already sufficiently developed but cone degeneration is not yet substantial, thus representing an all-cone retina. METHODS: Electroretinograms (ERGs) were obtained from 14 homozygous Rho(-/-) mice and eight C57Bl/6 control mice. The same individuals were tested every 7 days, beginning as early as postnatal day (P)14. The ERG protocols included flash and flicker stimuli, both under photopic and scotopic conditions. Retinal and choroidal morphology was observed in animals of comparable age. RESULTS: Functionally, the developmental phase lasted until postnatal week (PW)3 in both the Rho(-/-) mice and the control animals. During PW4 to 6, the Rho(-/-) mice showed a plateau in ERG parameters with normal or even supernormal cone responses and complete absence of rod contributions. At PW7, there was a marked onset of degeneration, which progressed so that no ERG signals were left at PW13, when the control eyes still had normal ERG responses. Microscopically, cone degeneration paralleled the functional changes, beginning at approximately PW6 and almost complete at PW13, whereas retinal pigment epithelium (RPE) and choroid did not show any abnormalities. CONCLUSIONS: From PW4 to 6, Rho(-/-) mice appear to have normal cone and no rod function. Despite the missing rod outer segment (OS), the structure of retina, RPE, and choroid remained unchanged. Therefore, the Rho(-/-) mice can serve during this age period as a model for pure cone function. Such a model is particularly useful to evaluate rod-cone interaction and to dissect rod- from cone-mediated signaling pathways in vivo.

AlphaB-crystallin in lacrimal gland duct and tears.

PURPOSE: To investigate alphaB-Crystallin expression and localization in the lacrimal gland and tear fluid. METHODS: Mouse, rat, porcine, monkey and human lacrimal gland samples were immuno-histochemically and immuno-electron-microscopically stained with various antibodies against alphaB-crystallin. Western- and Northern-blotting was performed to demonstrate the presence of alphaB-crystallin mRNA and protein. Human tear fluid was analyzed for the presence of alphaB-crystallin using dot blotting. RESULTS: alphaB-Crystallin is located in the lacrimal gland duct cells but not in the acini. Electron microscopically, the protein was frequently found in apical electron-dense granules of lacrimal duct cells, occasionally also in the duct lumina. Western blotting confirmed the presence of alphaB-crystallin in the lacrimal gland, Northern blot samples revealed the presence of alphaB-crystallin mRNA. In the human tear fluid, alphaB-crystallin was present in all samples investigated. CONCLUSIONS: We demonstrate for the first time that alphaB-crystallin is present in the lacrimal gland. Presence of the protein in apical secretory granules as well as presence in the tear fluid might indicate secretion of alphaB-crystallin into the tear fluid.

Efferent and afferent innervation of primate trabecular meshwork and scleral spur.

PURPOSE: To determine the correlation between nerve terminals and cells or extracellular matrix (ECM) components in different portions of the primate trabecular meshwork (TM) and scleral spur (SS). METHODS: Serial sagittal and tangential sections through the anterior segments of 10 cynomolgus monkey eyes and 12 human eyes were investigated immunohistochemically with antibodies against the vesicular acetylcholine transporter (VACHT), vasoactive intestinal polypeptide (VIP), tyrosine-hydroxylase (TH), neuropeptide Y (NPY), substance P (SP), calcitonin gene-related peptide (CGRP), and galanin (GAL) and with a reduced nicotinamide adenine dinucleotide phosphate-diaphorase (NADPHd) reaction. The distribution of the terminals was compared with that of alpha-smooth-muscle actin (SMA) staining in TM and SS. The relationship between terminals and adjacent cells or ECM components was also studied in ultrathin sections through the TM and SS of 11 monkey eyes cut in sagittal, tangential, and frontal planes. RESULTS: NADPHd-positive nerve terminals were present, especially in the outer portion of both human and monkey TM and in the SS. VACHT-immunoreactive (IR) fibers were found in human but not in monkey SS and TM. The fibers were most numerous in the elongated SS and posterior TM where most cells also stained for SMA. SP- and CGRP-IR nerve endings were also more numerous in the outer TM and SS than in the inner TM. Ultrastructurally, staining for SP was seen in nerve endings containing mitochondria and dense core vesicles and was in contact with the cribriform elastic network. In the posterior SS of monkey eyes were large terminals similar to those previously described in human eyes. CONCLUSIONS: The results show for the first time that in the primate TM and SS, there are cholinergic and nitrergic nerve terminals that could induce contraction and relaxation of TM and SS cells. Terminals in contact with the elastic-like network of the TM and containing SP-IR resemble afferent mechanoreceptor-like terminals in other parts of the body. These findings raise the possibility that the TM may have some ability to self-regulate aqueous humor outflow.

Induction of tissue transglutaminase in the trabecular meshwork by TGF-beta1 and TGF-beta2.

PURPOSE: To study whether human trabecular meshwork (HTM) cells are capable of expressing and secreting tissue transglutaminase (tTgase), an enzyme cross-linking extracellular matrix (ECM) proteins, and whether tTgase and synthesis of cross-linked fibronectin are increased after treatment of HTM cells with transforming growth factor (TGF)-beta1 or -beta2. METHODS: Anterior segments of six normal human eyes were stained with antibodies to tTgase. Tissues from three eyes were analyzed for tTgase using Western blot analysis. Monolayer cultures of HTM cells from eyes of five human donors were treated with 1.0 ng/ml TGF-beta1, -beta2, or 5 X 10(-7) M dexamethasone (DEX) for 12 to 96 hours. Induction of tTgase was investigated by Western and Northern blot analysis. External tTgase activity was measured by the ability to form polymerized fibronectin and the incorporation of biotinylated cadaverine into fibronectin. RESULTS: Labeling for tTgase was observed throughout the entire HTM. Cultured HTM cells expressed tTgase intra- and extracellularly. Treatment of cultured HTM cells with TGF-beta1 and -beta2 increased the tTgase mRNA and protein levels, whereas DEX had no effect. TGF-beta-treated HTM cells showed a significant increase in polymerized and unpolymerized fibronectin. Incorporation of biotinylated cadaverine was markedly increased when HTM cells were treated with TGF-beta for 24 hours before seeding. CONCLUSIONS: The enzyme tTgase is expressed in the HTM and is inducible by TGF-beta1 or -beta2 in cultured HTM cells. Extracellular tTgase is able to polymerize fibronectin. Increased levels of TGF-beta2 in the aqueous humor may lead to an increase of tTgase expression and activity in the HTM, causing an increase of irreversibly cross-linked ECM proteins. This mechanism might play a role for the increased outflow resistance seen in glaucomatous eyes.

Alpha-B-crystallin expression in tissues derived from different species in different age groups.

alphaB-Crystallin is constitutively expressed in a variety of tissues including the nervous system, the eye, heart and striated muscles and the kidney. The functional significance of the protein in the different cell populations is not yet known. Experimental data indicate that mechanical stress to the cells might play a role but that there is also a close correlation with markers of oxidative activity. Increased expression of alphaB-crystallin is seen in a number of age-related degenerative diseases. Whether aging per se induces expression of the protein has not been investigated yet. In this study tissue samples of the anterior eye segment, optic nerve, heart muscle and thyroid gland from mouse, rat, pig, cow and human donors of different age groups were investigated with immunohistochemical methods. alphaB-Crystallin levels in heart muscle and optic nerve samples from different species and different age groups were investigated using protein immunoblotting (dot blot) and the mRNA levels using semiquantitative PCR methods. The results showed that neither in heart muscle known to show constitutively high amounts of the protein nor in nonlenticular eye tissues with variations in staining intensity of different cell populations or in glandular cells studied for the first time, there were significant age-related staining differences. Dot blot methods as a quantitative evaluation method gave similar results. There were, however, species differences. In the eye these differences could be due to functional differences related to the development of a fovea centralis and an accommodative system in primates. In addition, in all mouse tissues there was less protein expression than in the other species. Differences in the absolute life span might be a factor involved in alphaB-crystallin expression. In summary the findings show that an increase in alphaB-crystallin with age may occur but is not a general phenomenon in tissues constitutively expressing this protein.

Vascular and glial changes in the retrolaminar optic nerve in glaucomatous monkey eyes.

Vascular and glial changes of the retrolaminar optic nerve were studied in monkey eyes with increased intraocular pressure (IOP) from 1 to 4 years and with different stages of optic nerve atrophy. In histological cross-sections of retrolaminar optic nerves of 11 rhesus and 6 cynomolgus monkeys the entire area, number of axons and vessels and area of pial septa were quantitated and three different kinds of nerve degeneration classified. Ultrathin sections of these different stages were performed and the number of open and occluded vessels was determined. In addition, in cynomolgus monkey optic nerves immunohistochemical staining for alphaB-crystallin, glial fibrillary acidic protein (GFAP) and vimentin was performed. Even in animals with the same duration of glaucoma and comparable mean IOP values the axon degeneration varied considerably. Independently of axon loss the number of capillaries in the rhesus monkeys remained constant, whereas there was a slight decrease in the cynomolgus monkeys. Some of the vessels, especially in the most severely damaged regions, were occluded. The density of glial cells increased whereas the total number remained nearly constant. In control sections all astrocytes stained for GFAP and alphaB-crystallin. In the glaucomatous optic nerves the density of alphaB-crystallin- and GFAP-positive cells was significantly increased. The vascular reaction in the retrolaminar glaucomatous optic nerves differs from that described in the prelaminar region. We assume that in the postlaminar region in areas with diminished nutritional needs vessels occlude and finally degenerate.