Heather Anderson

Chloroquine causes lysosomal dysfunction in neural retina and RPE: Implications for retinopathy

Chronic use of chloroquine has been shown to induce numerous pathophysiological defects in the retina. This drug has the ability to alter pH of intracellular compartments and lysosomal function of the retinal pigment epithelium (RPE) and retinal neurons may constitute the basis of chloroquine retinopathy. The aim of the current study was to investigate pathogenic alterations in retinal cells continuously exposed to chloroquine using appropriate in vivo and in vitro models. Male hooded Lister rats were implanted with osmotic mini pumps which released chloroquine continuously over a period of seven days. The eyes were processed for electron microscopy and ultrastructural abnormalities determined in the neural retina and quantified using stereology in the retinal pigment epithelium (RPE). RPE were also exposed to chloroquine in vitro and lysosomal pH changes were investigated using a pH sensitive probe. Degradative capacity was also analysed using FITC labeled rod outer segments (ROS). Chloroquine-treated animals displayed several ultra-structural abnormalities including numerous membranous cytoplasmic bodies (MCBs) in retinal neurons. Cone photoreceptors displayed numerous MCBs although rods did not. The RPE of the treated groups all showed significantly higher numbers of lysosomal associated organelles (LAO) than the control group (p < 0.001). The in vitro experiments demonstrated chloroquine-mediated rises in lysosomal pH and an increase in lysosome/phagosome accumulation of ROS in the chloroquine treated group (p < 0.01). The current study demonstrates that chloroquine disrupts lysosomal function in retinal neurons and RPE. The evidence presented provides a clear pathogenic basis for the functional defects experienced by patients with chloroquine retinopathy.

Diabetic retinopathy: quantitative variation in capillary basement membrane thickening in arterial or venous environments.

Diabetes mellitus was induced in male beagles by a single injection of an alloxan and streptozotocin cocktail and fasting blood sugar levels maintained between 15 and 20 mmol/l. Five years after induction of diabetes, three diabetic animals were sacrificed, together with sex and age-matched controls, and the retinas fixed for either transmission electron microscopy (TEM) or trypsin digestion. In TEM specimens, capillaries in close proximity to the major vessels were designated as either AE (arterial environment) or VE (venous environment) and the thickness of their basement membranes (BMs) measured using an image analyser based two dimensional morphometric analysis system. Results show that the BMs of retinal capillaries from the diabetic dogs were significantly thicker than those from control dogs. Furthermore, within the diabetic group the AE capillaries had thicker BMs than VE capillaries (p < or = 0.05). The controls, however, showed no significant difference in BM thickness between AE and VE capillaries. Although many of the capillaries designated as AE or VE would actually have been derived from the opposite side of the circulation, with respect to BM thickness, they conformed to values of their specific group. The conclusion is that diabetic capillaries are more vulnerable to BM thickening in an arterial environment than in a venous environment.

Fasciola hepatica: Tegumental changes induced in vitro by the deacetylated (amine) metabolite of diamphenethide

The effect of the deacetylated (amine) metabolite of diamphenethide (10 mugm/ml) on the tegument of Fasciola hepatica over a 24 hr period in vitro has been determined by means of transmission electron microscopy. In the tegumental syncytium, there is an initial accumulation of T2 secretory bodies at the apical surface (after 6 hr), together with increased exocytosis of secretory bodies and blebbing of the surface membrane. After 9 hr, the two surfaces of the fluke show different tegumental responses to drug treatment with a marked swelling of the basal infolds in the dorsal tegument, while the ventral tegument remains normal. By 18 hr, the swelling in the dorsal tegument is very severe, the entire basal region becoming edematous. In some areas, the tegument becomes detached to expose the basal lamina. The ventral tegument retains a fairly normal morphology, although there is a slight swelling of the basal infolds. The edema spreads internally to the cell bodies, beginning after 9 hr on the dorsal side of the fluke and 18 hr on the ventral side. By 18 hr, the flooding on the dorsal side is very severe and the cells attenuated, retaining few contacts with the surrounding parenchyma. From 9 hr onwards, there are progressive changes in cell structure, including a decrease in amount of granular endoplasmic reticulum and extent of its ribosomal covering, a decrease in numbers of secretory bodies, a swelling of the trans-most Golgi cisternae and disruption of the release of secretory bodies, and a swelling and disorganization of the mitochondria. The results are discussed in relation to the postulated activity of the deacetylated (amine) metabolite of diamphenethide as a Na+ ionophore.

The interaction between the deacetylated (amine) metabolite of diamphenethide (DAMD) and cytochemically demonstrable Na+/K+-ATPase activity in the tegument of Fasciola hepatica

The relative effects on tegumental Na+/ K+ -ATPase activity in Fasciola hepatica of the deacetylated (amine) metabolite of diamphenethide (DAMD) (10 Μg/ml, 18 h) and the Na+/K+-ATPase inhibitor ouabain (0.1 mM, 0.5 h) have been assessed cytochemically. In the normal tegument, Na+/K+-ATPase activity is particularly concentrated along the invaginations of the apical plasma membrane and the infoldings of the basal plasma membrane. Ouabain pretreatment significantly reduces the overall level of Na+/K+-ATPase activity, but does not induce swelling of the basal infolds. In contrast, DAMD does not significantly reduce either the level or distribution of Na+/K+-ATPase activity, but does cause a pronounced swelling of the basal infolds. The results are discussed in relation to the postulated action of diamphenethide as an inhibitor of Na+/K+-ATPase activity.

Fasciola hepatica: tegumental surface alterations following treatment in vitro with the deacetylated (amine) metabolite of diamphenethide

The effect of the deacetylated (amine) metabolite of diamphenethide (10 Μg/ml) on the tegumental surface of Fasciola hepatica over a 24 h period in vitro has been determined by scanning electron microscopy. Blebbing begins around the oral sucker after 3 h and then passes backwards along the body, reaching the ventral sucker and midbody by 6 h, and finally the posterior end of the body (by 12 h). Initially, the blebs are small, the tegument surrounding the spines is swollen and the tegument generally has a smooth, swollen appearance. This submerges the spines below the body surface. At higher magnification the surface is seen to bear microvillous-like projections in addition to the blebs and surface pitting is deeper than normal. Later on, the blebs increase in size and burst, causing lesions and loss of spines. Lesions begin to appear on the oral cone and ventral sucker after 6 h, in the midbody by 12 h and on the dorsal surface of the posterior region after 24 h. By this time the damage is extensive: around the oral and ventral suckers, and over large areas of the oral cone and midbody region the tegument has been stripped off to expose the basal lamina beneath. The dorsal surface of the fluke is consistently more severely affected than the ventral surface.

Diabetic retinopathy: morphometric analysis of basement membrane thickening of capillaries in different retinal layers within arterial and venous environments.

AIMS--To assess quantitatively variations in the extent of capillary basement membrane (BM) thickening between different retinal layers and within arterial and venous environments during diabetes. METHODS--One year after induction of experimental (streptozotocin) diabetes in rats, six diabetic animals together with six age-matched control animals were sacrificed and the retinas fixed for transmission electron microscopy (TEM). Blocks of retina straddling the major arteries and veins in the central retinal were dissected out, embedded in resin, and sectioned. Capillaries in close proximity to arteries or veins were designated as residing in either an arterial (AE) or a venous (VE) environment respectively, and the retinal layer in which each capillary was located was also noted. The thickness of the BM was then measured on an image analyser based two dimensional morphometric analysis system. RESULTS--In both diabetics and controls the AE capillaries had consistently thicker BMs than the VE capillaries. The BMs of both AE and VE capillaries from diabetics were thicker than those of capillaries in the corresponding retinal layer from the normal rats (p < or = 0.005). Also, in normal AE and VE capillaries and diabetic AE capillaries the BM in the nerve fibre layer (NFL) was thicker than that in either the inner (IPL) or outer (OPL) plexiform layers (p < or = 0.001). However, in diabetic VE capillaries the BMs of capillaries in the NFL were thicker than those of capillaries in the IPL (p < or = 0.05) which, in turn, had thicker BMs than capillaries in the OPL (p < or = 0.005). CONCLUSIONS--The variation in the extent of capillary BM thickening between different retinal layers within AE and VE environments may be related to differences in levels of oxygen tension and oxidative stress in the retina around arteries compared with that around veins.

Induction of alloxan/streptozotocin diabetes in dogs: a revised experimental technique

The diabetic dog represents an excellent model for use in many aspects of diabetic research. The present paper describes, in detail, a reproducible experimental protocol for the successful induction of chemical diabetes in beagles using a combination of the 2 pancreatic beta-cell cytoxic agents alloxan and streptozotocin.

FASCIOLA-HEPATICA - ULTRASTRUCTURAL-CHANGES TO THE TEGUMENT OF JUVENILE FLUKES FOLLOWING INCUBATION IN-VITRO WITH THE DEACETYLATED (AMINE) METABOLITE OF DIAMPHENETHIDE

Ultrastructural changes to the tegument of 5-week-old, 3-week-old and freshly-excysted Fasciola hepatica following in vitro incubation with the deacetylated (amine) metabolite of diamphenethide (DAMD, 10 microgramsml-1) were examined by transmission electron microscopy. A similar sequence of tegumental changes occurred in all three age groups of fluke, although, with increasing fluke age, the time before onset increased and the damage became more extensive. The 5-week-old flukes showed an initial stress response after 3 h, typified by blebbing of the apical plasma membrane, formation of microvilli and an accumulation and accelerated release of secretory bodies at the tegumental apex, as well as swelling of the basal infolds. The swelling increased in extent with progressively longer periods of incubation in DAMD, leading to extreme edema and sloughing of the tegument after 9 h. The 3-week-old flukes showed a stress response and swelling of the basal infolds after only 1.5 h, although sloughing of the tegument did not occur until after 9 h. In the freshly-excysted metacercaria, a stress response and some sloughing of the tegument were evident after only 0.5 h. At all stages of development, the ventral tegument was more severely affected than the dorsal. Changes also occurred to the tegumental cells which were indicative of a disruption in the synthesis and release of tegumental secretory bodies: the amount of GER became reduced, the cisternae became swollen and their ribosomal covering decreased, the Golgi complexes disappeared from the cells and the numbers of secretory bodies in the cells also decreased. The heterochromatin content of the nuclei increased and eventually the tegumental cells began to break down. Again, the changes became apparent more rapidly at the earlier stages of development. The ultrastructural changes to the tegument are linked to a possible mode of action for diamphenethide as an inhibitor of protein synthesis. In turn, the results may help to explain the drugs high efficacy against juvenile stages of F. hepatica.

Fasciola hepatica: Morphological changes in vitelline cells following treatment in vitro with the deacetylated (amine) metabolite of diamphenethide (DAMD)

Abstract Fasciola hepatica: morphological changes in vitelline cells following treatment in vitro with the deacetylated (amine) metabolite of diamphenethide (DAMD). International Journal for Parasitology18: 1061–1069. The effect of the deacetylated (amine) metabolite of diamphenethide (DAMD, 10 μg ml−1) on the vitelline cells of Fasciola hepatica over an 18 h period in vitro has been determined by transmission electron microscopy. DAMD acts preferentially on the undifferentiated stem cells and the intermediate cells in the early stages of protein synthesis; it appears to prevent their continued development. In the stem cell the nucleolus is absent after 6 h. During the rest of the drug treatment period considerable clumping of heterochromatin takes place, the cells round up and become electron-dense. Signs of autophagy are also evident, and the mitochondria become swollen and disorganized. From 6 h onwards there are progressive changes in the It1 (intermediate type 1) cells, including clumping of the heterochromatin in the nucleus, a decrease in the number of egg-shell globules (and consequently a reduction in the number and size of the shell globule clusters), and a decrease in the number of ribosomes on the GER cisternae, although the GER system remains well-developed. By 18 h the nucleolus is absent, and the cells are very rounded and electron-dense; the mitochondria are swollen and disorganized. Similar changes are evident in the It2 (intermediate type 2) cells, so that by 18 h it is difficult to distinguish between the It1 and It2 cells. In the mature cells there is a progressive decrease in the number and size of the shell globule clusters from 9 h onwards. Glycogen synthesis and ‘yolk’ formation may also be impaired and lipid droplets are present. Spaces begin to appear between the nurse cell cytoplasm and the vitelline cells after 9 h, and disruption of the nurse cell cytoplasm is evident after 12 h, becoming very severe by 18 h. By this time the follicle is very disorganized and empty-looking. In more severely affected follicles the mature cells are seen to be breaking down. Over the 18 h drug treatment period, a change in the cell population of the follicle takes place, with relatively more stem, early It1 and mature cells being present, whilst few if any characteristic It1 and It2 cells remain. The results are interpreted as being due to an inhibition of protein synthesis in the vitelline cells by DAMD.

Fasciola hepatica: Scanning electron microscopic observations of juvenile flukes following treatment in vitro with the deacetylated (amine) metabolite of diamphenethide (DAMD)

Abstract Five-week-old, 3-week-old and freshly-excysted Fasciola hepatica were incubated in vitro with the deacetylated (amine) metabolite of diamphenethide (10 μg ml −1 ), and the resulting tegumental surface changes examined by means of scanning electron microscopy. The damage which occurs follows a similar sequence in all three age groups. Thus, initially, the tegument becomes swollen, causing submergence of the spines. This is followed by the formation of blebs which increase in size and extent and eventually burst, leading to loss of spines, lesions and eventual sloughing of the tegument. With 5-week-old flukes, blebs are scattered over the entire ventral surface after 3 h, and loss of tegument begins in the ventral oral cone region after 6 h. By 18 h both dorsal and ventral surfaces are completely stripped of tegument. Large blebs occur on the ventral oral cone of 3-week-old flukes after only 1.5 h, lesions appear on the oral cone after 9 h, and by 18 h erosion of both surfaces is so severe that the fluke has begun to break up. Damage is extremely rapid in the case of the freshly-excysted flukes, with tegument having been stripped off most of the ventral surface after only 1 h, and by 2 h little recognizable structure remains. Therefore, susceptibility to diamphenethide in vitro decreases with increasing age of the fluke; this observation agrees with in vivo efficacy studies.