In Vitro

In vitro cultures of HTM cells were firstly examined in medium only conditions to

investigate whether cells derived from donors of different ages exhibited any

differences in morphology or cytoskeletal arrangement. Any differences observed in the current cohort may then be inferred as being an age-related feature of cells. There was minimal variation apart from a small decline in CLAN incidence with increasing donor age but it was not considered strong enough to merit a true pattern. Grouping of donors into old and young revealed there was no statistically significant difference, thus confirming the trend. The age range available included tissue from infants under 1 year old and extended to 91 years old. There is limited literature exploring

differences of TM cell in vitro that have been derived from different donors never mind the potential influence of donor age. Studies using foetal TM cells suggested they make a good model as they have similar ultrastructural features as adults (Lee et al., 2008). It would seem then that the percentage of CLAN containing cells under control conditions at least is influenced solely by the in vitro environment.

Given that TFG-β2 was shown to induce CLAN formation in earlier experiments it was also utilised here. As before treatment with 2ng/ml TGF-β2 induced CLANs above medium only levels, however, the induction was variable with the percentage of CLAN containing cell ranging from 9% to 40%. CLAN incidence was found to have some correlation with donor age but there were 4 donors in which the percentage of CLANs did not appear to fit with the pattern of the age range. In an attempt to explain these anomalies cultures were re-evaluated morphologically. In 2 young donors (027 aged 5

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cultures contained cells of a predominantly epithelioid nature even in medium only conditions. In some areas of these cultures a cobblestone-like effect was described with the actin localising to the cell periphery as observed in corneal endothelial cells (Gordon, 1990). Contrasting these results were the strangely low CLAN counts obtained in donors 009 and 010 aged 58 and 80 years respectively. The presence of TGF-β2 resulted in values of 9%, when the same treatment in most other older donors had produced values of 20-37%. Morphological inspection of these cells noted that while they were epithelioid in the presence of TGF-β2, the cells were very spindle shaped and highly aligned in medium only cultures. Even with the TGF-β2 induced change in cell shape the main cytoskeletal arrangement was straight stress fibres. During the re-evaluation of HTM cell shape it was noted that the only other culture above infancy to be reported as highly spindle in medium was 020 aged 36 years. In the presence of TGF-β2 15% of cells were found to contain a CLAN. With donors 009 and 010 excluded this was the lowest CLAN incidence above 10 years old. All other donors above 10 years old were described as being predominantly cigar shaped or epithelioid in medium only conditions.

It has been observed that 3 distinct cell types exist in the outflow pathway; endothelial cells from schlemm’s canal, TM cells from the uveal and corneoscleral and TM cells from the cribriform region (Flugel et al., 1991), each with its own primary function (Alvarado et al., 2004). Rohen et al (Rohen et al., 1982) suggested that TM cell cultures derived from explants were likely mainly derived from cribriform layer. If the cells of a particular culture had a different ratio of cell types this might account for some of the difference observed.

Another explanation for the differences observed may be provided by the evaluation of cell shape in relation to donor age. The majority of HTM cells from younger donors were more spindle in shape and could compact close together while cells from older donors tended to be more epithelioid. Experiments using both TGF-β2 and DEX

revealed that cell shape is highly indicative of actin arrangement, with epithelioid cells more likely to contain a CLAN.

By removing the 4 out layers based on morphological differences the linear correlation between CLAN incidence and age was much stronger (R2=0.49). The division of the donors into young and old revealed that in the presence of 2ng/ml TGF-β2 older donors produced significantly more CLANs than younger donors. The increase in the percentage of CLAN containing cells in presence of TGF-β2 was not the only value in which we were interested. It has been stated here that TGF-β2 induced CLAN formation above medium only conditions in all donors regardless of age. When the percentage induction was presented in graphical form it was clear that two distinct groups were visible with young donors having consistently lower CLAN incidence than older donors. The linear regression between donor age and percentage of CLAN induction was 0.58 and when taken together we felt positive that TGF-β2 induces CLAN formation in a greater number of HTM cells from older donors than younger donors. Although this was an extensive study, increasing the number of donors involved may help to cement the trends observed. Special attention to the middle section is most certainly needed, as only 2 donors within the current study fall

between 10 and 50 years (23 and 36 years old). Both were closely associated with the much younger donors than the older donors when percentage induction was assessed, however, the percentage of CLAN containing cells in medium and TGF-β2 were

variable (donor aged 23 had high CLAN incidence in medium only and TGF-β2 while donor aged 36 had lower than expected values).

As to why HTM cells from older donors are more susceptible to TGF-β2 induction we have examined some of the processes related with ageing in the TM. With increasing age there is a decreased number of TM cells (Alvarado et al., 1981; Grierson and Howes, 1987) which has been attributed to apoptosis (Agarwal et al., 1999; Baleriola et al., 2008; Sibayan et al., 1998). Others have shown that the level of senescence (Liton et al., 2005) is also increased in vivo. Increased incidence of senescence and apoptosis are linked to stresses such as ROS. If older cells are already compromised then the addition of TGF-β2 may be sufficient to increase CLAN incidence. In chapter 6 we deal with several age-related changes which have been reported in TM cells,

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