Biomedical subjects

G F Vrensen

Publications and source records attributed to G F Vrensen.

At least 55 records · Page 3Linked to original sources

PubMed1996

Sympathetic innervation of the rat choroid: an autoradiographic tracing and immunohistochemical study.

The sympathetic innervation of the choroid was investigated by means of the anterograde tracer 3H-leucine, injected into the rostral part of the superior cervical ganglion. The tracer was autoradiographically visualized at the light- and electron-microscopic levels. Labelled unmyelinated fibres were found in the choroid and labelled terminals were observed in close proximity to the smooth muscle cells of arterioles. The labelled terminals contain granular vesicles of different size and clear vesicles; the mitochondria were of the electron-dense type and no cell-to-cell synaptic contacts were observed. In addition, tyrosine hydroxylase immunocytochemistry was performed. Immunostained terminals found in the choroid show ultrastructural features similar to those found in the tracing experiments: granular vesicles of different size, clear vesicles and electron-dense mitochondria. On the basis of previous studies and the present observations, it is concluded that the choroid has a dual innervation, a parasympathetic innervation from the pterygopalatine ganglion and a sympathetic innervation from the superior cervical ganglion. The dual innervation is most likely involved in maintaining the homeostasis of vasodilatation and vasoconstriction in the choroid.

Animals↗

PubMed1996

Calcium distribution in the human eye lens.

The distribution of calcium in the human eye lens was investigated using the oxalate-pyroantimonate technique. In epithelial cells and superficial cortical fibers calcium is sequestered and tightly bound to proteins in the endoplasmic reticulum, nuclear envelope, Golgi cisterns and mitochondria. In intermediate fibers these cell organelles are broken down and the liberated calcium is ionically bound to the phospholipids at the extracellular side of the fiber-limiting membrane. It is argued that calcium is probably additionally bound to beta-crystallin. This site-specific variation in calcium storage might explain the deeper cortical localization of early focal dot opacities and retrodots.

Adult↗

PubMed1996

Epithelial cell-fibre coupling in the equator of the human lens.

A detailed freeze fracture study of human lenses of various ages revealed that coupling between epithelial cells and fibres occurs only in a limited area in the equator of the lens; it also demonstrated that the extent of coupling is independent of age. The developing fibres remain coupled to their original neighbours and no other contacts between epithelial cells and fibres appear to be established.

Cell Membrane↗

PubMed1996

Aging affects the conformation of cholesterol in the human eye lens.

The distribution of 3-beta-OH-cholesterol along the optical axis of 20 human eye lenses of different ages was measured using confocal Raman microspectroscopy in combination with the 3-beta-hydroxysteroid-specific probe filipin. This non-destructive technique allow a spatially very detailed study of the distribution of 3-beta-OH-cholesterol in individual lenses. It revealed age-related changes of the 3-beta-OH-cholesterol distribution along the optical axis. The 3-beta-OH-cholesterol was found to be distributed asymmetrically along the optical axis and this distribution was found to change with age. The relative 3-beta-OH-cholesterol content in the anterior part of the lens decreases significantly with increasing age compared with its equal posterior counterpart. Additionally all the lenses have a very low 3-beta-OH-cholesterol content in the central part of their nuclear region, the area where the primary lens fibres are located.

Adolescent↗

PubMed1996

Ultrastructural organization of human corneal nerves.

PURPOSE: Although the human cornea is densely innervated, observations of the nerve fiber distribution and ultrastructure are scarce. This study aimed to provide a detailed electron microscopic analysis of nerve fibers in the central and peripheral human cornea. METHODS: Samples from seven fresh corneas, obtained from the eyes of persons with melanoma, were processed for light and electron microscopic examinations. Both frontal and cross-sections were studied. Furthermore, serial ultrathin sections from the mid-epithelium to the anterior stroma were used. RESULTS: Unmyelinated nerve fiber bundles (as many as 30 nerve fibers and cross-section as large as 20 micrometers) run parallel to the stromal collagen fibers. Nerve fibers contain clear, dense cored and dense vesicles and are ensheathed by thin rims of Schwann cell protrusions and amorphic matrix. Some nerve fibers invaginate the cytoplasm of keratocytes. After passing through Bowman's membrane, bundles of straight fibers (cross-section 0.1 to 0.5 micrometers) and single-beaded nerve fibers, which both lack Schwann cell ensheathment, run parallel in an alternating manner. Beaded nerve fibers, containing many mitochondria and glycogen (cross-section as large as 2 micrometers), turn upward and invaginate both basal and wing cells. Except for the presence of myelinated nerve fibers in the peripheral stroma, no differences in the central cornea were observed. CONCLUSIONS: Nerve fibers invaginating epithelial cells and keratocytes suggest that both cell types are directly innervated. The presence of vesicles, mitochondria, and glycogen in stromal and epithelial nerve fibers suggest that classical and peptidergic transmitters, probably of sensory origin, innervate the human cornea. Peptidergic transmitters in nerve fibers may be involved in neuroimmunomodulation of the cornea.

Aged↗

PubMed1995

Retrograde fluorescent microsphere tracing of retinal and central afferents to the nucleus of the optic tract in pigmented and albino rats.

The pretectal nucleus of the optic tract is involved in optokinetic nystagmus. Albino animals suffer from inverse optokinetic nystagmus, possibly due to a smaller number of ipsilateral projections. Animals with laterally placed eyes have an asymmetric nystagmus, attributed to a presumed lack of binocular cortical input to the nucleus of the optic tract. Moreover, a direct input from the ipsilateral retina is controversial. We investigated the retinal and central afferents to the nucleus of the optic tract in the rat by retrograde tracing using fluorescent latex microspheres. Ipsilaterally projecting retinal ganglion cells were observed in pigmented and also in albino rats. Projections from the visual cortex and primary visual nuclei were demonstrated.

Animals↗

PubMed1995

Efferent projections of the olivary pretectal nucleus in the albino rat subserving the pupillary light reflex and related reflexes. A light microscopic tracing study.

The olivary pretectal nucleus is a primary visual centre sensitive to luminance changes. It is involved in the pupillary light reflex, the consensual pupillary light reflex and related reflexes, such as the lid closure reflex whereby pupillary constriction takes place. Since the olivary pretectal nucleus is a small nucleus, previous studies using degeneration, horseradish peroxidase and radioactive amino acid tracing were limited regarding to the exclusiveness of the projections from the olivary pretectal nucleus. In the present study the position of the olivary pretectal nucleus in the rat was first localized by physiological recording of the neurons upon luminance stimulation. Subsequently, an anterograde tracer Phaseolus vulgaris leucoagglutinin was injected iontophoretically. This allows a much more precise localization of the olivary pretectal nucleus projections. Ascending and descending pathways originating from the olivary pretectal nucleus were observed. Ascending fibres project bilaterally to the intergeniculate leaflet, the ventral part of the lateral geniculate nucleus and ipsilaterally to the anterior pretectal nucleus. In addition, contralateral projections were observed to the zona incerta and the fields of Forel. Descending fibres project bilaterally to the periaqueductal gray, the nucleus of Darkschewitsch, the interstitial nucleus of Cajal, the Edinger-Westphal nucleus and the intermediate gray layer of the superior colliculus. Also a contralateral projection to the oculomotor nucleus and an ipsilateral projection to the pontine nucleus and the nucleus of the optic tract were found. Furthermore, the contralateral olivary pretectal nucleus received a small projection. Retrograde tracing experiments using two fluorescent dyes revealed that the fibres projecting to the contralateral olivary pretectal nucleus and to the contralateral interstitial nucleus of Cajal are collaterals. The projection from the olivary pretectal nucleus to the facial nucleus which has been described to receive an input in cats could not be confirmed for the rat. The fact that the Edinger-Westphal nucleus, the interstitial nucleus of Cajal and the superior colliculus receive an input from the olivary pretectal nucleus suggests that this primary visual centre is not only involved in the pupillary light reflex, but also in controlling eye and head position and saccadic eye movements. Although visual acuity largely depends on receptive field sizes of retinal ganglion cells and their central connections, the stronger sympathetic influence during the pupillary light reflex in animals with frontally placed eyes compared to animals with laterally placed eyes may also contribute to the higher visual acuity in animals with frontally placed eyes.

Afferent Pathways↗

PubMed1995

Efferent synaptic organization of the olivary pretectal nucleus in the albino rat. An ultrastructural tracing study.

In this study an ultrastructural analysis was made of the efferent projections of the olivary pretectal nucleus in the rat. The anterograde tracer Phaseolus vulgaris leucoagglutinin was injected iontophoretically into the olivary pretectal nucleus. Ascending and descending pathways were studied. In the descending pathway special attention was paid to the fine structural features of the olivary pretectal nucleus efferents projecting to the Edinger-Westphal nucleus, the interstitial nucleus of Cajal, the nucleus of Darkschewitsch and the periaqueductal gray. The projection to the superior colliculus and the pontine nucleus was also studied at the ultrastructural level. All the labeled terminals in the descending pathway showed ultrastructurally similar features: clear, round vesicles and electron dense mitochondria. The terminals made asymmetric synaptic membrane specializations (Gray type I), the postsynaptic profiles were dendritic. In the interstitial nucleus of Cajal and the superior colliculus the terminals are organized in glomerulus-like structures. The terminals in the descending pathway were enwrapped by astrocytic processes, also in the glomerulus-like structures. In the ascending pathway the projection to the ventral part of the lateral geniculate nucleus was studied. Almost all terminals in the ascending pathway showed similar ultrastructural features as in the descending pathway: electron dense mitochondria, clear, round vesicles and asymmetric synaptic membrane specializations (Gray type I). The terminals are organized in glomerulus-like structures. To identify the projecting neurons in the interstitial nucleus of Cajal and the Edinger-Westphal nucleus, retrograde tracing experiments were performed. Therefore the beta subunit of cholera toxin conjugated with horseradish peroxidase was injected into the facial nucleus.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1995

Aging of the human eye lens--a morphological point of view.

Age-related cortical lens changes are mainly due to oxidative stress induced membrane disorganization increasing with time to (1) membrane ruptures, (2) local fibre opacification, (3) local opacities and eventually to (4) more advanced segmental and annular opacities and mature cataract. Early occurrence of these cortical opacities is delayed by the presence of several defence systems: (1) radical scavengers, (2) a membrane repair-lysosomal system, (3) a segregation system and (4) the inertness of the deep cortical and nuclear membranes. Posterior subcapsular age-related changes As shown by Spector (1991), oxidative stress also affects the DNA of lenticular epithelial cells leading to single strand breaks. Although these DNA breaks are efficiently repaired by excision repair, longlasting low dose insults, as in the human lens or massive insults as in experimental studies, can lead to incomplete repair and therefore to germinative epithelial cells with aberrant DNA. These will give rise to cohorts of deviating cells with an abnormal developmental programme. That abnormal DNA, e.g. regarding the genes for cytoskeletal proteins, leads to abnormal development of fibres has been shown in studies in transgenic mice (Dunia et al., 1991). Irradiation in animals leading to abnormal development of fibres and finally to posterior subcapsular cataract has been extensively documented (Worgul, 1989). In our studies of donor eye lenses, we have incidentally found lenses with one or two regions with abnormal and abnormally interdigitating lens fibres which would beyond doubt have led to posterior subcapsular cataract. Compared with the other two types of age-related changes, posterior subcapsular changes are rather infrequent (Sasaki et al., 1987, 1989).(ABSTRACT TRUNCATED AT 250 WORDS)

Aging↗

PubMed1995

Light and electron microscopic findings on experimental melanomas after hyperthermia at 50 degrees C.

The effect of hyperthermia, 50 degrees C applied for 5 min, on the development of lesions in hamster Greene melanomas was investigated. Hyperthermia was induced by a laser that produced radiation at 780-880 nm. Hamster melanomas were also examined after arrest of the blood circulation to differentiate between heat-induced lesions and those caused by ischaemia due to vascular occlusion. Tumours were removed 5 and 30 min and 1, 3, 6 and 24 h after thermotherapy. The cytotoxic effects of heat and ischaemia were examined by light and electron microscopy. Hyperchromatic nuclear staining, the first hyperthermia-induced lesion, was detected in the superficial layers of the tumour 5 min after heat treatment. The lesions had progressed to severe pyknosis and extended into deeper layers of the tumour 1 h after hyperthermia. At 24 h necrosis was observed at a depth of 6 mm. The hyperthermia-induced lesions differed markedly from the ischaemia-induced lesions, in that the latter showed early mitochondrial damage but nuclear pyknosis did not become manifest until 3 h after arrest of the blood circulation.

Animals↗

PubMed1995

Maturation of fiber membranes in the human eye lens. Ultrastructural and Raman microspectroscopic observations.

Electron microscopic, freeze fracture and cholesterol cytochemical studies have revealed that the maturation of lens fibers is accompanied by considerable changes in the ultrastructure of their limiting membranes. These changes are in line with biochemical studies on the cholesterol content in the lens and support the physiological notion that upon maturation lens membranes are transformed from normal permeable to nonleaky membranes. In addition it is shown that early cortical changes involved disturbances of lens membranes. As a consequence it can be postulated that, in order to be significant, future studies on the early events leading to cataractous disturbances must be carried out using a combination of histochemical, immunohistochemical and regional biochemical approaches.

Anti-Bacterial Agents↗

PubMed1995

Novel aspects of the ultrastructural organization of human corneal keratocytes.

PURPOSE: Proper functioning of the endothelium and proper structural organization of the keratocytes and collagen bundles are of ultimate importance for transparency of the cornea. The role of the endothelium has been investigated extensively, whereas the role of the keratocytes is still unclear. Detailed knowledge on the ultrastructural organization of keratocytes and the relationship between keratocytes and collagen bundles is as essential for understanding corneal transparency as is knowledge of endothelial functioning. METHODS: Thirty-five corneas (30 postmortem donor corneas and 5 fresh corneas from the operating theater; age range, 28 to 90 years) were used for light microscopy, transmission electron microscopy, and scanning electron microscopy. Serial frontal sections of the central stroma reaching from epithelium to endothelium and cross-sections were studied. At three levels, reconstructions of the mutual arrangement of keratocytes were made using semithin sections. RESULTS: Keratocytes have the appearance of highly active cells with an abundancy of organelles. Between the dendritic ramifications of these cells, large amounts of amorphous material is observed. One of the most remarkable observations is the presence of an extensive network of fenestrations along the surface of the keratocytes. Another important observation is the circular arrangement of keratocytes gradually turning clockwise like a corkscrew from epithelium to endothelium. CONCLUSIONS: From the current study, the following conclusions can be drawn: Keratocytes are not quiescent but are highly active cells probably involved in turnover of the extracellular matrix; fenestrations may be of functional relevance with respect to facilitation of diffusion and mechanical attachment of the collagen fibers to the keratocytes; the corkscrew organization of keratocytes suggests that they form completely closed sheets of communicating cells throughout the depth of the cornea, creating equal chances for all light rays to pass one or more keratocytes and thus minimizing variation in light scattering over the entire cornea.

Adult↗

PubMed1995

Calcium localization and ultrastructure of clear and pCMPS-treated rat lenses.

PURPOSE: The sulfhydryl complexing agent p-chloromercuri-phenylsulfonate (pCMPS) has been shown to increase lens membrane permeability, Na+ and Ca2+ content, and light scatter in the rat lens in vitro. This study aimed to investigate the ultrastructural changes accompanying the increase in light scatter. In addition, high-resolution histochemistry was used to study the cellular distribution of Ca2+ in normal and cataractous lenses. METHODS: Rat lenses were incubated for 4 hours in normal (1 mM) and high (5 mM) Ca2+ containing media supplemented with 40 microM pCMPS. Control lenses were incubated in 1 mM Ca2+ containing medium. They were prepared for scanning and transmission electron microscopy and for Ca2+ localization using the oxalate-pyroantimonate procedure. RESULTS: Control lenses incubated for 4 hours had normal morphology and showed no evidence of light scatter. Calcium distribution as observed with the oxalate-pyroantimonate precipitation method was low in superficial fibers, high in the membranes of intermediate fibers, and declined again toward the nucleus. In the deeper cortex, there also were small vacuoles of calcium accumulation. pCMPS treatment (in 1 and 5 mM Ca2+) induced a significant influx of calcium into the lens cytoplasm. Calcium-containing extracellular vacuoles also were seen in the intermediate cortex in both cases. The presence of these vacuoles appeared to correlate with the major areas of light scatter in the lens. In 5 mM Ca2+, intracellular vacuoles were observed throughout the superficial cortex. CONCLUSIONS: Most of the calcium observed by oxalate-pyroantimonate in the normal lens is located at the membrane, and the staining appears strongest in the intermediate cortex. In pCMPS treatment, large extracellular vacuoles are present in this intermediate zone and appear to be the major source of light scatter. This zone may be the initiation site of many different types of cataract, including some described in human lenses.

4-Chloromercuribenzenesulfonate↗

PubMed1995

New approach to assess the cholesterol distribution in the eye lens: confocal Raman microspectroscopy and filipin cytochemistry.

Confocal Raman microspectroscopy (CRM) is a non-invasive, non-destructive, and sensitive analytical tool for the study of some aspects of the molecular organization of cells and tissues with high spatial resolution. Filipin, a polyene antibiotic, specifically binds to cholesterol, and its molecular structure predicts it to be Raman-active. The aim of the present study was to assess the potentialities of a combined CRM-filipin approach to study the distribution of cholesterol in the human eye lens. Paraformaldehyde-fixed human lenses were sliced (0.7 mm), incubated with filipin, and analyzed by CRM. Filipin proved to give a specific Raman signal at 1586 cm-1, hardly interfering with signals from lens proteins. The CRM-filipin approach proved to be extremely sensitive, allowing detection of cholesterol in the femtogram range. It has an excellent spatial resolution (0.2-0.5 micron 3) when using point measurements. Due to the intrinsic anisotropy of membranes in the eye lens and therefore of the cholesterol distribution, a line-scan approach has to be adopted when fiber-to-fiber changes in cholesterol are of interest. The distribution of filipin along the optical axis of four human eye lenses was compared with data from the literature. The combined CRM-filipin approach is a highly specific and sensitive method for the study of cholesterol within cells and tissues. The spatial resolution is high and can be adapted to the desired discriminative power. The gross distribution of filipin along the optical axis obtained in this study is similar to that found in biochemical studies.

Cholesterol↗

PubMed1994

Combined elastic and Raman light scattering of human eye lenses.

The distribution of the scattering coefficient (as defined in the appendix) at a wavelength of 647.1 nm along the visual axis of human eye lenses was investigated using a specially designed set-up for spatially resolved measurements of the intensity of the scattered light. For the same lenses, the distribution of the protein content was measured using confocal Raman microspectroscopy. Data collected by both methods were processed in terms of a recently developed theory of short-range, liquid-like order of crystallin proteins that accounts for eye lens transparency. Seven fresh intact human lenses of varying age have been investigated. In addition, elastic and Raman scattering measurements have been performed on fixed lenses. The main results and conclusions are: (1) Fixation significantly affects the light scattering properties of the eye lens. The average level of scattering increases and a change in the distribution of scattered light intensity along the visual axis occurs. Protein content and average distribution were not altered by fixation. (2) There are significant differences between the distribution of the scattering coefficient for lenses of different ages. For young lenses (18 and 20 years) regions with a low protein content (anterior and posterior cortex) show a higher level of elastic light scattering, while for older lenses (42-78 years old) there is no obvious correlation between the scattering level and protein content. (3) Changes in the level of light scattering along the visual axis of the lens cannot be explained by protein concentration effects.(ABSTRACT TRUNCATED AT 250 WORDS)

Adolescent↗

PubMed1994

Blue-light-induced dysfunction of the blood-retinal barrier at the pigment epithelium in albino versus pigmented rabbits.

The purpose of this study was to determine the role of epithelial melanin in blue light phototoxicity of the retina. The first manifestation of the phototoxicity has been shown to be a breakdown of the blood-retinal barrier at the retinal pigment epithelium. The blood-retinal barrier function of six New Zealand albino rabbits was compared to that of four pigmented chinchilla rabbits after exposure to broad-band blue light (400-520 nm). Additionally, the spectral sensitivity of blood-retinal barrier dysfunction was determined by exposing 15 New Zealand albino rabbits to narrow-band blue light with peak intensity at lambda = 408 nm, 418 nm, 439 nm, 455 nm and 485 nm (bandwidth: 11.7-13.5 nm). The blood-retinal barrier function was evaluated with vitreous fluorophotometry. Ultrastructural changes and permeability of the retinal pigment epithelium for horseradish peroxidase were evaluated in the albino rabbits with electron microscopy. Exposure to broad-band blue light up to 832 J cm-2 demonstrated the blood-retinal barrier of albino and pigmented rabbits to be equally sensitive. Electron microscopy of albino rabbits exposed to above-threshold energy demonstrated an increase of inclusion bodies in the retinal pigment epithelium and vacuolation of the cytoplasm. Transcellular passage of intra-arterially administered horseradish peroxidase through the pigment epithelium into the subretinal space was seen. The narrow-band exposures demonstrated that light of 439 nm was more effective than the light of other wavelengths in inducing barrier dysfunction in albino rabbits. This implies that chromophores absorbing at 439 +/- 6 nm were responsible for the phototoxicity in albino rabbits. The results indicate that melanin does not have a damaging nor a protective role in phototoxicity since (1) the presence of melanin is not essential for blue-light-induced photochemical damage to the blood-retinal barrier at the retinal pigment epithelium, and (2) protection from this sort of damage is not greater in melanin containing epithelia than in non-melanin containing epithelia.

Animals↗

PubMed1994

Peripheral neural circuits regulating IOP? A review of its anatomical backbone.

The peripheral nervous system is classically separated into a somatic division containing both afferent and efferent pathways and an autonomic division composed of efferents only. The somatic afferent division is divided in A- and B-neurons. The B-neurons are supposed to be autonomic afferents as part of a reflex system involved in homeostasis. Recent data obtained by neuronal tracing and immunohistochemical experiments concerning the eye related peripheral nervous system endorse the existence of these peripheral reflex systems. Somatic afferents of trigeminal origin synaptically innervate parasympathetic neurons in the pterygopalatine ganglion. This probably represents a pathway mediating autonomically regulated ocular activity in response to sensory stimulation. In addition, it has been hypothesized that trigeminal sensory nerve fibres have an efferent function in response to noxious stimuli e.g. the ocular injury response. Sympathetic nerve fibres originating in the superior cervical ganglion course through the trigeminal and pterygopalatine ganglion without forming direct synaptic contacts. These fibres, however, contain clusters of vesicles suggesting some kind of interneural communication. Parasympathetic nerve fibres of pterygopalatine origin course through the ciliary ganglion. These nerve fibre terminals also contain clusters of vesicles without direct synaptic contacts. Experimental data concerning the distribution of neuropeptides revealed a more detailed knowledge of the anterior eye segment innervation. These experimental data are subject to some debate. The pros and cons of different techniques are discussed. Neural circuits regulating IOP have long been postulated. The possible role of peripheral reflex systems in the regulation of IOP is discussed.

Anterior Eye Segment↗

PubMed1994

Basic fibroblast growth factor stimulates corneal endothelial cell growth and endothelial wound healing of human corneas.

PURPOSE: To determine the dose response of human recombinant basic fibroblast growth factor (bFGF) on mitogenic activity, and the supplementary role of serum in cultured bovine and human corneal endothelial cells (BCECs, HCECs). To investigate the effect of bFGF on endothelial wound healing of human corneas in vitro. METHODS: In cell culture, DNA synthesis was assessed by 3H-thymidine incorporation. Wound healing was studied using paired human corneas after mechanical damaging of the endothelium. One cornea was treated with bFGF, and the mate served as control. Wound closure was determined after staining with trypan blue. Endothelial cell density (ECD) was assessed in the closed wound area after alizarin red staining. DNA synthesis was assessed using 3H-thymidine autoradiography. RESULTS: In cell culture, bFGF induced a dose-dependent mitogenic response on BCECs and HCECs. Addition of serum to the culture medium shifted the dose-response curve to considerably lower bFGF concentrations. In organ culture, the time of complete wound closure shortened only marginally (0.5 day) after bFGF treatment (P < 0.01). In the closed wound center, ECD was significantly higher in 1 ng/ml bFGF-treated corneas (686 +/- 134 cells/mm2) than in controls (554 +/- 117 cells/mm2), an increase of +25%. Doses of 0.1 and 10 ng/ml also were effective, but less so than with 1 ng/ml (+11% and +15%, respectively), whereas a dose of 100 ng/ml even had a negative effect (-11%). DNA synthesis was marginally enhanced in bFGF-treated (1 ng/ml) corneas. CONCLUSIONS: The maximal effective dose of bFGF producing a BCEC mitogenic response is dependent on serum. In human senior donor corneas, bFGF promotes endothelial wound healing predominantly by stimulation of cell migration.

Aged↗