CLINICAL AND ANALYTICAL STUDIES IN POSTMENOPAUSAL WOMEN SYMPTOMATIC OF DRY EYE
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Introduction Menopause which is defined as a permanent physiological, or natural, cessation of menstrual cycle, plays an important role in the development of ocular surface dryness symptoms and there is an increased prevalence of dry eye in women, especially those aged over 50. Despite the high prevalence of dry eye in post-menopausal women (PMW), very few studies have been undertaken to understand dry eye disease in a group of PMW who are not on Hormone Replacement Therapy (HRT). Studies in the past on PMW have primarily focused on the relationship between HRT and dry eye. Hence, a series of studies were undertaken to understand the clinical aspects of dry eye and their relationship to a variety of tear film components, in a group of PMW with and without symptoms of dry eye. The specific aims of each chapter were as follows: • Chapter 4: To characterize symptoms of dry eye using questionnaires, namely Ocular Surface Disease Index Questionnaire© (OSDI) and the Indiana Dry Eye Questionnaire (DEQ). • Chapter 5: To characterize clinical signs and symptoms in participants who present with and without symptoms of dry eye. • Chapter 6: To compare tear osmolality and ferning patterns in participants with and without dry eye symptoms. • Chapter 7: To investigate the potential relationship between subjective symptoms and clinical signs with tear film lipocalin and lysozyme concentrations in participants with and without dry eye symptoms. • Chapter 8: To optimize a technique for the isolation of total RNA (ribo nucleic acid) and total protein derived from conjunctival epithelial cells collected via conjunctival impression cytology (CIC). • Chapter 9: To quantify the expression of MUC1 (mucin1) and MUC16 (mucin16) mRNA and protein and to investigate the potential relationship between mucin expression and tear film breakup time in a group of participants with and without dry eye symptoms. Methods • Chapter 4: Participants were categorized as being symptomatic or asymptomatic of dry eye based on their response to the OSDI questionnaire. These results were then compared to the DEQ, which has questions related to the frequency of ocular surface symptoms and their diurnal intensity. • Chapter 5: Non invasive tear breakup time (NITBUT) was evaluated using the ALCON Eyemap®. Tear volume was assessed using the Phenol Red Thread (PRT) test and bulbar conjunctival hyperemia was measured using objective (SpectraScan PR650© Spectrophotometer) and subjective (slit lamp) methods. • Chapter 6: Tears were collected via capillary tube. A freezing point depression osmometer was used to measure the osmolality of the tear film. The tear ferning test was performed and evaluated for the quality of ferning, based on the Rolando grading system. • Chapter 7: Tears were collected via capillary tube and an eye wash method. Tear lysozyme and lipocalin concentrations were determined via Western blotting. • Chapter 8: CIC was collected using either Millipore (MP) or Poly Ether Sulfone (PES) membranes. RNA and protein isolation was performed using two different RNA isolation techniques. Two methods of protein isolation from CIC discs were evaluated. RT-PCR of mRNA for MUC1 and western blotting of lipoxygenase type 2 protein (LOX2) was performed to confirm the collection of intact RNA and total protein respectively. • Chapter 9: Tears were collected via capillary tube and an eye wash method. CIC was collected using MP membrane. Expression of MUC1 and MUC16 mRNA was assessed via real time PCR. Expression of both membrane-bound and soluble MUC1 and MUC16 were quantified via Western blotting. Results • Chapter 4: The OSDI total score and sub scores for the Non Dry Eye (NDE) and Dry Eye (DE) groups were significantly different (NDE =7.43 ± 7.71 vs DE = 24.87 ± 13.89; p<0.001). The DEQ scores showed that the DE group exhibited a higher frequency and intensity of symptoms than the NDE group, which worsened as the day progressed (p<0.001). • Chapter 5: The DE group exhibited a significantly shorter NITBUT (5.3 ± 1.7 vs 7.0 ± 2.7 secs; p=0.0012). Tear volume was significantly lower for the DE group (19.3 ± 5.1mm vs. 16.3 ± 5.6mm; p=0.031). Bulbar hyperemia was significantly higher in the DE group for both objective (u’ = 0.285 ± 0.006 vs. 0.282 ± 0.006; p=0.005) and subjective techniques (48.4 ± 10.0 vs 40.6 ± 10.4; p=0.0011). • Chapter 6: Osmolality values in DE individuals were significantly higher than the NDE (328.1 ± 20.8 vs. 315.1 ± 11.3 mOsm/kg; p = 0.02). There was a significant difference between the DE and NDE participants for the ferning patterns (p = 0.019). No significant correlation between tear osmolality and tear ferning was noted (DE: r = 0.12; p > 0.05, NDE: r = -0.17; p > 0.05). • Chapter 7: No difference in tear lysozyme or lipocalin concentration was found between DE and NDE groups, irrespective of tear collection method. Method of collection significantly influenced absolute concentrations (p<0.008). • Chapter 8: There was no significant difference between the two procedures used to isolate RNA and protein from CIC membranes (p>0.05). Total RNA yield was greater with the MP membrane. The mean yield of protein extracted from MP membrane using the two protein isolation techniques also did not show a significant difference. • Chapter 9: No difference was found in the expression of either MUC1 or MUC16 protein or mRNA expression between symptomatic DE and NDE (p>0.05). Weak correlations were found between the NITBUT values compared with either soluble or membrane bound MUC1 and MUC16 expression. Conclusions • Chapter 4: Questionnaires are useful tools to symptomatically divide participants into dry eyed and non dry eyed candidates. However, the questionnaire used to categorise patients can impact on the outcome variables determined. • Chapter 5: Post-menopausal women with dry eye symptoms demonstrate shorter NITBUT, lower tear volume and increased bulbar conjunctival hyperemia than those who have no symptoms. • Chapter 6: Tear osmolality in DE is higher than in NDE. There is a tendency towards less ferning in persons over 50 years of age, regardless of their symptoms. • Chapter 7: Comparison of clinical data with lipocalin and lysozyme concentrations failed to reveal statistically significant correlations. The concentration of either protein was not associated with tear stability or secretion. • Chapter 8: The total RNA yield was greater with the MP membrane. RNeasy Mini (RN) (Qiagen) method is recommended due to enhanced speed as well as on-column isolation and DNase digestion capabilities. CIC with MP membranes followed by immediate freezing and then extraction and processing facilitates the collection of total protein from human conjunctival cells. • Chapter 9: No difference was found in the expression of either MUC1 or MUC16 protein or mRNA expression between symptomatic PMW and asymptomatic controls.