ESTUDIO BÁSICOS DE INGENIERÍA

Neural retina

Optic nerve

Inferior collection of

subretinal fluid

Removal of epiretinal

membrane

Subretinal fluid

Fig. 2.8. Diagrams to show the sources of subretinal fluid; from an eye with rhegmatogenous retinal detachment (a) and from an eye with proliferative vitreoretinopathy (b; after Tarrant, 1986 and with the kind permission of Dr.P.Hiscott).

Patient Number

Sex Age Diagnosis Sample

Number 1 F 64 Longstanding retinal detachment with PVR (Grade C) (Dose response curve) 2 M 47 Traumatic longstanding retinal detachment RDI 3 M 26 Recurrent rhegmatogenous retinal detachment RD2 4 M 68 Rhegmatogenous retinal detachment RD3 5 M 45 Longstanding retinal detachment with PVR (Grade C) PVRl 6 M 55 PVR (Grade C) PVR2 7 F 21 Proliferative diabetic retinopathy PDRl 8 M 24 Proliferative diabetic retinopathy PDR2 9 F 70 Proliferative diabetic retinopathy PDR3 10 M 40 Proliferative diabetic retinopathy PDR4

Fig. 2.9. Table of patient and diagnostic details of SRF samples used in the migration studies.

tested for their migration to each of the treated RCE aliquots with untreated RCE as thecontrol.

Finally media conditioned by the three cell types in culture was employed in migration and preliminary characterisation studies as well as settlement and proliferation assays, its preparation and use is described in sections 2.4.1. and 2.4.2. respectively.

2.3.3. Zigmond Hirsch Chequerboard Analysis of the Migratory Response

The cellular migratory response in vitro has several components including chemotactic and chemokinetic movement (see section 1.3.2.). Fibronectin has been shown to stimulate both chemotaxis and chemokinesis of fibroblasts (Postlethwaite et al, 1981; Joseph et al, 1987). To be able in the future to prevent the migration of the cells to the site of ERM formation in PVR, it is necessary to understand fully the types of migratory response of the appropriate cells so that they may be blocked. Therefore in order to characterise the migratory response of the cells Zigmond Hirsch chequerboard analysis was employed (Zigmond et al, 1973; Postlethwaite et al, 1981).

The Zigmond Hirsch chequerboard analysis involves adding different concentrations of chemoattractant above and below the membrane in different wells and counting the number of cells migrated to the lower side of the membrane for each well. In the wells where there is no chemoattractant (Fig. 2.10a.) only a minimal number of cells are found on the lower side of the membrane possibly due to the combined effects of random locomotion and gravity. Where the chemoattractant has been added only to the upper wells (Fig. 2.10b.), the larger proportion of cells will be on the same side of the membrane as the stimulant and there is no concentration differential across the membrane to attract the cells through the pores and so any stimulated random movement taking place in the upper well where the chemoattractant is to be found should not involve movement through the pores. When the chemoattractant is in the lower wells (Fig. 2.10c.) if chemotactic movement is taking place then the cells move up a concentration gradient of stimulus and the cells pass through the pores in the membrane to the underside where the chemoattractant is located. In wells where the concentration of

chemoattractant is the same above and below the membrane (Fig. 2.10d.) there is no gradient and if large numbers of cells pass through the pores it is by stimulated random movement

(chemokinesis).

In a 48-well modified Boyden chamber, a series of four different concentrations of stimuli (including zero) were tested. They were run in quadruplicate by using two chambers (two groups on each chamber). The test substances were added to the bottom wells in a vertical direction and the same concentrations were repeated at right angles in the upper wells (see Fig. 2.10e.). This resulted in a diagonal series of wells where the upper and lower concentrations of test substance was equal and there was no gradient across the membrane. The other intervening wells had varying degrees of positive and negative gradients. Each experimental run to assess chemotaxis and chemokinesis using the Zigmond Hirsch chequerboard was run at least three times.

2.3.4. Mixed Labelled Migration of the Three Cell Types

There is little clear information in the literature as to which of the three cell types is the most effective at responding to migratory signals. It would of course be of considerable value to know which of the cell types is the most responsive because it may well further our understanding of the genesis of ERMs. As the chemotactic response is receptor dependent, the migration of the cells seems to be very largely reliant on their condition and treatment during the assay. Slight variations in the culture conditions and state of the cells may alter their migratory response and as the Boyden chamber assay is miniaturised it would be more susceptible to any environmental variations and this may result in noticeable variations in the numbers of migrated cells. In order to eliminate some of these variables the cell types were migrated under as near identical conditions as possible.

It was decided to develop a system which would truly compare the migratory rates of the test cells by migrating them together in the same chemotaxis well. It was felt that as well as being more analogous to the in vivo situation, mixed migration was a natural progression from the classic "one cell type" chemoattraction studies. To migrate the three cell types together it was necessary to label the cells so that they

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