biochemistry of retinal dystrophy · in contrast, hmp shunt activity in dystrophic retinae is...

Journal of Medical Genetics (1970). 7, 277.

Biochemistry of Retinal DystrophyH. W. READING*

The study of progressive hereditary degenerationof the retina in laboratory animals in conditions re-sembling human retinitis pigmentosa was one ofProfessor Arnold Sorsby's major research interestsduring the past three decades. It was in this field ofwork that I was privileged to be his collaborator fora number of years. The search for a biochemicallesion as the causative agent in such conditions wascarried out in the M. R. C. Wernher Research Unitfor Ophthalmological Genetics, in London, andencompassed detailed histopathological and bio-chemical investigations. In addition, the experi-mental production of similar lesions using specificretinotoxic substances was pursued with greatvigour in the hope that knowledge of the pharma-cological mode of action of such compounds wouldoffer some means of counteracting their toxic effects.The ultimate aim of such an approach was the pro-vision of therapeutic measures to combat or offsetthe retinal degenerative process in human retinitispigmentosa (Sorsby, 1941; Noell, 1953; Edge et al.,1956; Sorsby and Nakajima, 1958a, b; Sorsby andHarding, 1962a, b; Sorsby and Reading, 1964;Reading and Sorsby, 1966b; Reading, 1970).

It would be impracticable to review all the workdone in all laboratories on this problem, so it is in-tended in the present paper to describe and reviewthe biochemical and histochemical studies on retinaldystrophies in animals other than man. In addition,an attempt is made to correlate present knowledge togive a rational analysis of the sequential nature of thebiochemical lesion which undoubtedly underlies theprogress of these genetically determined retinaldegenerations.

HistopathologyKeeler (1927, 1928a, b) was the first to record

the finding of a strain of 'rodless' mice in which thevisual cells were almost completely absent. Bruck-ner (1951) presented the first ophthalmoscopicdescription of the fundus of albino and pigmented

* Address: Medical Research Council, Brain Metabolism Unit,Department of Pharmacology, University of Edinburgh, 1/2 GeorgeSquare, Edinburgh EH8 9JZ.

wild mice affected with retinal dystrophy. Theincidence was as high as 50% in some of the strainsexamined.

Sorsby et al. (1954) carried out histological in-vestigations on Bruckner's mice, and established thatthe degeneration process in the retina developedsubsequent to cellular differentiation but before thefinal stages of development of the rods. Similarchanges were observed in the rat by Bourne,Campbell, and Tansley (1938) and by Lucas, Att-field, and Davey (1955), in the Irish setter by Lucas(1954), and in other breeds of dog by Barnett (1966).In affected mice, retinal development is normal untilaround the 10th postnatal day, whereupon earlydegenerative changes are seen. In the rat and dog,retinal degeneration starts about 12 days after birth,though full differentiation of the rods does notoccur until 21-28 days. The pattern of changes inthe rat broadly resembles those occurring in humanretinitis pigmentosa (Cogan, 1950).The condition was shown to have a simple auto-

somal recessive inheritance in the three speciesmentioned. Sorsby et al. (1954) stressed the factthat affected retinae were degenerate before theneuroepithelium had reached the stage of fulldifferentiation. They suggested the term dys-trophy rather than abiotrophy to describe such dis-orders.

Electron MicroscopyLasansky and De Robertis (1960) examined the

dystrophic mouse retina by electron microscopy,confirming the failure of the full development of therod cells, and showed that the outer segment of therod in the normal mouse proceeds from a primitivecilium to the formation of membranous materialwhich is reorientated into sacs, finally building upinto a definite regularly layered structure. In thedystrophic animals, changes occur in the reorienta-tion stage and the regularly layered structure neverappears; in its stead, degeneration sets in. Dowlingand Sidman (1962), in a similar examination of theeyes of rats affected with retinal dystrophy, reportedan overproduction of visual pigment associated withthe appearance of swirling sheets of 'extracellular

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lamellae' lying between the outer segments of therods and the pigment epithelium. These authorsalso described the electroretinogram (ERG) patternin the dystrophic rat and showed that it developedalmost to the normal adult state but thereaftershowed abnormalities.

Biochemical InvestigationsVirtually all the biochemical investigations have

been carried out on retinae or eyes of pink-eyedpiebald agouti rats descended from the originalstrain of Bourne et al. (1938).

Carbohydrate metabolism. The dependenceof the retina on carbohydrate as a fuel for the supplyof energy prompted early investigators of retinaldystrophy to direct their attention towards studiesof retinal glucose metabolism. Graymore, Tansley,and Kerly (1959) found anaerobic glycolysis in theaffected retina to be virtually the same as in the nor-mal retina until the third postnatal week. Theseworkers were unable to establish a relation betweenanaerobic glycolysis and visual cell degeneration.Walters (1959) and Brotherton (1962) reported adecrease in the rate of anaerobic glycolysis inaffected retinae, but this was not pronounced until16-25 days of age.Aerobic glucose metabolism was investigated in

detail using radioisotopic methods by Reading andSorsby (1962). The normal animals used for com-parison purposes in these experiments were black-hooded (PVG) rats, a strain characterized by anabsence of spontaneous retinal degeneration. Theage range studied was from 10 to 60 days. Nostriking differences between normal and affectedretinae were found in the over-all pattern of glucosemetabolism from 10 to 21 days after birth. At laterstages, dystrophic retinae showed obvious decreasesin lactic acid, carbon dioxide, and amino acid pro-duction, together with respiratory depression and afall in the rate of the utilization of glucose. Dys-trophic retinae did not retain the amino acids formedfrom glucose intracellularly; in fact the amino acids'leaked' into the fluid medium bathing the tissue.This occurred before the animals had reached theage of 21 days.

Protein and amino acid metabolism. Leak-age of amino acids suggested possible changes inprotein metabolism in the affected retina, so the up-take, transport, and incorporation of '4C glycineinto total retinal protein as a measure of the over-allrate of protein synthesis was studied both in vitroand in vivo (Reading and Sorsby, 1964a, b). Ex-periments were carried out on litter mates of affected

rats in comparison with normal animals at corres-ponding ages from 6 to 24 days after birth. Invitro, retinal tissue was incubated for 2-hour periodsin a phosphate medium with added glucose anduniformly labelled 14C glycine. In vivo, each ratreceived a subcutaneous injection of 10 jLCi of 14Cglycine in saline. Retinal protein was fractionatedby tricholoracetic acid precipitation followed byremoval of nucleic acid and lipids. The isolatedprotein fractions were then assayed for radioactivity.

In vitro, glycine incorporation into retinal proteinwas lower in dystrophic than in normal retinae. At6 to 8 days of age the differences were most pro-nounced (p = 0 04) and became less with increasingage, until at 24 days, incorporation rates were equalin both normal and dystrophic animals (Fig. 1).

180 Normal retinae180 p=004 1ZI Dystrophic retinae

160 0, 4'p>O02

z 140

2k 120-C-

E i. I 1 1 °- =0*

80-

0-0~~~~~~~~~~~=-

40-

6 8 10 12 16 24Age (days)

FIG. 1. Rate of retinal protein synthesis (in vitro). (From Bio-chemistry of the Retina, p. 78. Ed. by C. N. Graymore. AcademicPress, London and New York. 1965).

No differences in amino acid uptake or in transportof amino acid into retinal tissue were found be-tween normal and affected retinae at the early stages.Only at 24 days was the intracellular concentrationof glycine less in dystrophic than in normal tissue.The in vitro results were confirmed by measuringthe rate of retinal protein synthesis at variousintervals after the subcutaneous injection of radio-active glycine into live 8-day-old litter mate rats.The dystrophic animals showed a conspicuous de-crease in the rate of protein synthesis and a reduc-tion in the rate of protein breakdown, indicating aslower turnover of retinal protein. The rates ofprotein synthesis in the livers of both normal and

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Biochemistry of Retinal Dystrophy

affected rats were equal, so the differences observedin the retinae were unlikely to be due to differencesbetween the animal strains. In addition, the ratesof amino acid transport into the retinae were thesame in normal and dystrophic animals, in vivo.The depression in protein synthesis and turnover

was apparent some 6 to 8 days before histologicalchanges were detectable. This suggested a dis-crepancy in the production of an essential protein,possibly one with structural as well as functionalproperties. The anomaly could involve either areduction in total protein or the production of aprotein of slightly abnormal structure.

Hexose monophosphate shunt (HMP) path-way. These findings prompted an investigation ofbiochemical mechanisms associated with the visualcycle. The HMP pathway of direct glucose oxida-tion has been implicated in the normal functioningof the visual cycle in the mammalian retina by theelegant researches of Futterman (1963). After lightfalls on the retina causing bleaching and the releaseofthe chromophore (all-trans retinal) from the visualpigment, the reduction of retinal to retinol (vitaminA alcohol), by the specific retinal alcohol dehydro-genase (RADH), is dependent on an adequatesupply of the reduced pyridine nucleotide coenzyme,NADPH. This enzymatic reduction by RADHinvolves the oxidation of NADPH to NADP, soproducing an increase in the cytoplasmic NADP/NADPH ratio. This results in a stimulation ofglucose oxidation via the HMP shunt pathwaywhich requires NADP for its two dehydrogenases.In this way, the two processes are 'metabolicallycoupled' by the common coenzyme. In the normalretina, the HMP shunt shows a low level of activityin dark adaptation, with short bursts of activitywhen light falls on the retina.The activity of the HMP shunt pathway was in-

vestigated in developing normal and dystrophic ratretinae (Reading, 1964). This was done in vitroby measuring the incorporation of 14C into res-piratory carbon dioxide produced by incubatingexcised retinal tissue with specifically 14carbon-1 or14carbon-6 labelled glucose substrates. The resultsobtained showed an increase in HMP shunt activityin dystrophic retinae. In normal, undifferentiatedretinae at 6 to 8 days of age, HMP shunt activity isrelatively high, the activity of this pathway decreas-ing as development proceeds. In contrast, HMPshunt activity in dystrophic retinae is approxi-mately twice that of the normal at correspondingages, during the early stages of development of thelesion (Table I). Though the statistical signifi-cance of the results is of a low order, the trend to-

TABLE IACTIVITY OF HEXOSE MONOPHOSPHATE SHUNT

IN NORMAL AND DYSTROPHIC RETINAE

Age (dy.) Normal Dystrophic Increase inDystrophic

6-7 2-9 (±+06) 41 (±+08) 410 (p=0 3-0 2)14 1-7 (±03) 2-5 (+05) 490 (p=02-0-1)27-28 1-2 (+0-1) 1-8 (+0 2) 50-0 (p = 0 05 - 0 02)

(SEM in parentheses.)Results expressed as ratios of specific yields of 14Co2 from 14C-1

glucose, and 14C-6 glucose.(From Reading, 1964.)

wards an increased activity is obvious. Bonavita(1965) gave confirmatory evidence to the study ofover-all HMP shunt activity described above. Hedetermined the specific activities of the two pri-mary enzymes of the HMP shunt pathway, viz.glucose-6-phosphate dehydrogenase (G6PD) and6-phosphogluconate dehydrogenase (6PGD) innormal and dystrophic rat retinae. Bonavitafound decreases in the activities of both G6PD and6PGD during development and differentiation inboth normal and affected retinae. However, asignificant difference was found between normal andaffected animals in that G6PD specific activityreached a peak value in dystrophic retinae at 12days of age (about 60% higher than normal), andthis decreased to reach the values of normal retinae,both values then remaining about equal up to 120days of age. A similar pattern was shown by6PGD (i.e. a peak activity at about 12 days), thoughlower values were found in dystrophic retinae at allstages of development.

Retinal alcohol dehydrogenase (RADH)activity. The enzyme RADH is metabolicallycoupled to the HMP shunt in the retina, as pre-viously explained. Since changes in the latter path-way had been found, it was thought that there mightbe a reflection of these manifested by early changesin RADH activity in dystrophic retinae. A studyof RADH activity carried out by Reading andSorsby (1966a) showed that the enzyme increasedin activity in normal retinae until the animals were 1month old, and then levelled off to reach a steadystate of activity in the adult (Fig. 2). In dystrophicretinae, RADH enzyme activity developed similarlyuntil the rats were about 2 weeks old, whereuponwide variations in activity were recorded. Subse-quently an obvious decrease in activity was foundfrom 3 weeks onwards, until retinae from 4-week-olddystrophic rats possessed only 40% RADH activityof corresponding normal retinae. Decrease inRADH activity in dystrophic retinae was therefore

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350

300

j._

.-1 250.'

u

ov

0 c

V -

'U.:k

50

.

0

0~

0

0

A

0 5 10 I5 20 25 30

Age (days)5

monthsFIG. 2. Retinal alcohol dehydrogenase activities in developingnormal and dystrophic rat retinae. 0-0 normal retinae; A-Adystrophic retinae. (From Reading and Sorsby (1966a. )

not obvious until after the third week of life. At 18to 20 days of age, there was virtually no differencein enzymatic activity between normal and affectedretinae. At this stage, the dystrophic animal showssubstantial degenerative changes in the rod outersegments. It was concluded from these experi-ments that the decrease in RADH activity could beregarded as consequential to the primary bio-chemical lesion which causes cellular degeneration.

Isoenzyme changes in retinal dystrophy.Futterman and Kinoshita (1959) subjected extractsof bovine retinae to zone electrophoresis on starchpaste and assayed isolated fractions for lactic de-hydrogenase (LDH) activity. They found fivediscrete fractions, all with the capability of con-

verting pyruvate to lactate, but having differentaffinities for the pyridine nucleotide coenzymes,NADH and NADPH, which depended on substrate(pyruvate) concentration.

Bonavita, Ponte, and Amore (1963) reported onlyfour distinct fractions from the rat retina, but Gray-more (1964a, b) using cellulose acetate strips as thesupporting medium for electrophoresis, was able toshow five fractions from rat retina. The fivefractions consist of two major and discrete frac-tions, the 'M' (muscle) and 'H' (heart) isoenzymes,while the remaining three are hybrids of these. It

is thought that the 'M' isoenzyme is concernedmainly with anaerobic glycolysis (lactate formation)and the 'H' isoenzyme associated with highly oxy-genated tissues is inhibited by high concentrations ofpyruvate. Tissues with predominantly 'H' typeLDH tend to oxidize pyruvate via the Krebs cyclerather than convert it to lactate, so the retina ischaracterized by having a predominantly 'M' typepattern. Bonavita et al. (1963) and Graymore(1964a, b) both showed an anomalous isoenzymepattern for LDH in dystrophic rat retinae. Theyfound the 'M' type to be deficient from birth.Graymore suggested that the 'M' fraction played animportant role in the differentiation process in theretina.

Anomalies in visual pigment formation.Dowling and Sidman (1962), in their extensiveinvestigation of retinal dystrophy in the rat,demonstrated an overproduction of visual pigmentin this condition. This was shown by electronmicroscopy and by rhodopsin analysis of the backportion of the eye which included retina, pigmentepithelium plus choroid and sclera. As alreadystated, the excess rhodopsin was found in the formof swirling sheets or bundles of extracellular lamellaehaving a double membrane structure which, thoughbeing disorganized, resembled the general ap-pearance of normal outer segments under the elec-tron microscope. In fact, the authors describedthis as the first recognizable abnormality. Theystated that by the age of 20 days, dystrophic animalspossessed about twice as much rhodopsin per eye ascorresponding controls. Rhodopsin content in-creased rapidly in both affected and normal eyesuntil about 30 days of age, when the dystrophic eyecontained almost double the amount of rhodopsinas the control. Subsequently, rhodopsin content inthe eyes of dystrophic rats fell rapidly and by 40 daysthe level was about halfthat ofthe normal. Evidencewas also presented that decreases in rhodopsin con-tent, the onset of histological changes, and changesin the ERG of the dystrophic eye could be delayedby keeping the animals in the dark from birth.

Vitamin A metabolism. The distribution ofretinal (vitamin A aldehyde) and retinol (vitamin Aalcohol) when compared in normal and dystrophiceyes under the conditions of dark and light adapta-tion were found to differ in the two strains (Reading,1966). Litter mates 1 to 4 weeks old were light ordark adapted and killed by cervical fracture. Afterenucleation, retinae were separated from the tissuesof the back of the eyes (referred to as 'pigmentlayers' for convenience). In the dark adaptation

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experiments manipulations were carried out underthe illumination of a red darkroom safelight. Thetissues were assayed for both retinol and retinal.

In normal animals, dark adaptation producedsubstantial increases in retinal content of retinae,while light adaptation (bleaching) caused decreasesin retinal content; changes that were to be expected.However, it was found that prolonged bleaching,even when the retina had reached a state of func-tional maturity at 4 weeks, never reduced the level ofretinal to below 11-0-12 0 ,ug./g. (wet wt.) tissue.Conversely, in dystrophic animals, bleaching pro-duced a conspicuous depletion of retinal in retinae.In addition, dark adaptation in dystrophic animalsresulted in the regeneration of less visual pigmentthan normal, and this process decreased with in-crease in age (Fig. 3).

_? Normal Affected

1-m. 14

> 12i 100 8.u

o 4).42

0

Normal

IH [1 n2

Aqe (weeks)

Affected

.n n-

H3 4 1 2 3 4

FIG. 4. Retinol (vitamin A alcohol) content of the 'pigment layers'from eyes of normal and dystrophic rats after light adaptation.

TABLE IIAVERAGE WET WEIGHTS OF RAT RETINAE AND

PIGMENT LAYERS (in mg.)

Retinae Pigment LayersAge(wk.)

Normal Dystrophic Normal Dystrophic

1 3-7 2-8 7 9 6-32 6-3 5-6 6-3 8 83 9 0 7-9 10-0 10-94 11-8 12-7 10-0 11-4

FIG. 3. Retinal content of normal and dystrophic rat retinae underconditions of light and dark adaptation.

The most striking differences between normal anddystrophic animals were seen in the retinol contentof the 'pigment layers' at 2, 3, and 4 weeks of age.

In the eye of the dystrophic rat, concentrations ofretinol after light adaptation were at least twice thosein the normal rat at corresponding ages (Fig. 4).The figures obtained must have been subject tosome dilution owing to the presence of excess tissue(choroid and sclera), so that the absolute differencescould have been greater than those recorded. Thewet weights of retinae and 'pigment layers' fromnormal and dystrophic animals showed a remarkablesimilarity over the age-group examined (Table II).

Changes in ATPase. The implication ofATPase in Na + and K + transfer in cell membranesand the changes reported by Dowling and Sidman(1962) in the ERG at 22 days, coinciding with de-generation of rod inner segments and nuclei in the

dystrophic rat, prompted Bonavita, Guaneri, andPonte (1966) to compare the development of activityof Na + -K+ -activated ATPase in normal and dys-trophic rats. They found that Mg+ + -activatedand Mg + + -Na + -K + -activated ATPase activitiesincreased during postnatal development of the retinain both normal and affected rats. However, a de-cline in Na + -K + -ATPase activity could bemeasured much earlier than 22 days after birth.These authors expressed their results in the formof the ratio Mg + +Na +K I/Mg + + stimulatedATPase activity. In this way they found a pro-nounced decrease in the ratio by 12 days of age indystrophic retinae. In normal retinae, this ratioremained constant over the period 2 to 120 days ofage.

Discussion of Present State of KnowledgePrimary biochemical lesion. The most im-

portant biochemical findings recorded up to thepresent are first, the apparent overproduction ofvisual pigment in the eyes of dystrophic rats com-pared with normal-sighted animals at correspondingages (Dowling and Sidman, 1962), and secondly,the excessive local concentration of retinol (vitaminA alcohol) found in the 'pigment layers' of dystro-phic rats, occurring before the onset of histologicalchanges (Reading, 1966).

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These two observations indicate that there is ex-cessive production of visual pigment of an unusuallylabile nature, labile, that is, to the action of light,since affected rats reared in darkness develop visualcell degeneration much more slowly than thosereared under normal lighting conditions.

Dowling and Sidman (1962) stated that the excessvisual pigment was qualitatively normal, judged bythe wavelengths of the peaks of its absorption spec-trum and those of the products of bleaching.Caution must be attached to any interpretationbased on this observation, however, since the spectralcharacteristics simply show that the 'dystrophicvisual pigment' contains the normal chromophore(1 1-cis retinal) and that the protein moiety (opsin)has a structure similar in molecular size and aminoacid composition to that present in the normal un-affected eye. The protein moiety of the 'dystro-phic pigment' could possess a relatively minoranomaly in one of its constituent peptide chains,viz. one or more amino acids 'out of place'. Such asituation accords with the genetic nature of thecondition, and suggests reasons why the visual pig-ment of the dystrophic animal is excessively labileto the action of light.The most recent ideas concerning the breakdown

of visual pigment (rhodopsin) and the propagationof the nerve impulse have been put forward byBonting (1969). Bonting and his associates work-ing at Nijmegen have obtained good experimentalevidence to support their ideas which are based onthe known constitution of rhodopsin, i.e. a complexof protein (opsin), phospholipids (e.g. phosphatidyl-ethanolamine), and retinal. They regard thevisual mechanism as a process in which the rho-dopsin present in the outer segments of the rod cellsabsorbs a quantum of light energy and causes firsta steroisomeric change of the chromosphore from1 1-cis to all-trans retinal. The all-trans retinalthen moves from its normal bonded position in therhodopsin molecule (i.e. forming a Schiff base withthe amino group of phosphatidylethanolamine) tothe E-amino group of lysine, a basic amino acidconstituent of the protein opsin. This E-aminogroup carries a positive charge and during its com-bination with retinal, this positive charge is lost.This opens up a channel for the passage of Na + andK + ions in the outer rod sac membrane, and socationic fluxes occur that result in the propagationof the nerve impulse. In the regeneration process,the normal cationic gradient with high Na + con-centration on the outside and high K + concentra-tion on the inside of the membrane is re-establishedby the Na+ -K + -ATPase present in the rod sacmembrane, the substrate ATP being supplied from

the inner segment of the rod cells. It is tempting tospeculate that if the amino acid sequence of opsinproduced under the influence of an abnormal alleleis slightly altered from the protein normally pro-duced, then the changes described above may occurwith greater or less facility. Not only must theamino acid structure of the protein be important,but also the conformational nature or the tertiarystructure of the protein must be maintained withinvery close limits, since the protonation of one par-ticular group in one amino acid (lysine) situated in aspecific position in the peptide chain is of paramountimportance to the proper functioning of the over-allmechanism.

Sequential nature of lesion. It is obviousfrom the foregoing discussion that very little can bedone to prevent the development of the primarybiochemical lesion. However, the series of changesthat occur and finally result in the breakdown anddisappearance of the entire neuroepithelium of theretina must involve some sequential biochemicalmechanisms. It appears that the excessive localconcentration of retinol in the pigment epithelium(Reading, 1966) arises from the action of light on anunusually labile type of visual pigment; in fact, a'dystrophic type rhodopsin' itself present in abnor-mally large amounts in the early stages of the con-dition. The concentration of vitamin A to main-tain tissue integrity, especially that of the retina andits associated layers in the eye, is critical. VitaminA deficiencies, produced in rats and rabbits by re-placing vitamin A alcohol with vitamin A acid in thediet, show obvious types of retinal degeneration(Dowling, 1964; Sorsby, Reading, and Bunyan,1966).The build-up of retinol in the pigment epithelium

will tend to cause breakdown of the membranes ofthe subcellular lysosomes in this tissue, thus re-leasing bound acid proteases and hydrolases. Thisaccounts for the cellular digestion and subsequentdisappearance of cellular debris. Preliminary evi-dence has been found for the release ofacid cathepsins(proteases) into the cytoplasm of retinal cells fromdystrophic rats. Such changes do not occur in corre-spondingnormalrats (H. W. Readingand M. Burden,unpublished results). So far it has been impossibleto estimate such changes in the pigment layers owingto the limits of sensitivity of the methods of estima-tion. Though the evidence for the appearance ofthe protease in the soluble cytoplasm of the retinacells is unequivocal, it is difficult to judge the stageat which such enzymatic release could have a patho-logical effect. Supporting evidence for the releaseof lysosomal enzymes has recently been provided by

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elegant cytochemical studies on the eyes of dys-trophic rats (Yates et al., 1970). In these studiesacid phosphatase appears to be released in the pig-ment epithelium and probably permeates the outersegments of the rod cells. In addition, the earlyrelease of a lysosomal acid protease has also beendemonstrated.The evidence for the release of lysosomal en-

zymes offers some hope of alleviating or offsettingthe development of retinal degeneration in animals,possibly including man. It may be possible toblock some of the sequential stages in the neuro-pathological process. Pharmacologically activeagents are known which stabilize lysosomal mem-

branes, such as cortisol and cortisone (Allison,1968), just as other substances, such as excess vita-min A, disrupt the membrane or render it permeable(Dingle and Lucy, 1965).

In this way, the future holds some hope for thedirect application of the basic researches which havebeen carried out on inherited retinal dystrophies;researches that were instituted and pursued withgreat drive by Arnold Sorsby. It appears at thepresent moment that a cytochemical approach offersthe best means of attacking the unanswered ques-

tions. This approach would eliminate the diffi-culties inherent in obtaining sufficient quantities ofeye tissue to show significant changes, which hasalways been the major problem for biochemists andothers working on retinal dystrophy. It needhardly be stated that the experimenter must alwaysrely on tissues obtained from experimental animals.

The author expresses his gratitude to Miss Joan M.Pittendreigh for her careful and painstaking typing ofthe manuscript.

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biochemistry of retinal dystrophy · in contrast, hmp shunt activity in dystrophic retinae is...

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