David L. Easty

Age related compliance of the lamina cribrosa in human eyes

AIMS To investigate changes in the mechanical compliance of ex vivo human lamina cribrosa with age. METHODS A laser scanning confocal microscope was used to image the surface of the fluorescently labelled lamina cribrosa in cadaver eyes. A method was developed to determine changes in the volume and strain of the lamina cribrosa created by increases in pressure. The ability of the lamina cribrosa to reverse its deformation on removal of pressure was also measured. RESULTS Volume and strain measurements both demonstrated that the lamina cribrosa increased in stiffness with age and the level of pressure applied. The ability of the lamina cribrosa to regain its original shape and size on removal of pressure appeared to decrease with age, demonstrating an age related decrease in resilience of the lamina cribrosa. CONCLUSIONS The mechanical compliance of the human lamina cribrosa decreased with age. Misalignment of compliant cribriform plates in a young eye may exert a lesser stress on nerve axons, than that exerted by the rigid plates of an elderly lamina cribrosa. The resilience of the lamina cribrosa also decreased with age, suggesting an increased susceptibility to plastic flow and permanent deformation. Such changes may be of importance in the explanation of age related optic neuropathy in primary open angle glaucoma.

Conclusions of the corneal transplant follow-up study

AIM On the basis of finalised data from the Corneal Transplant Follow up Study to identify and quantify factors influencing corneal graft outcome in terms of graft survival, rejection, visual acuity, and astigmatism. METHODS Multifactorial analysis of 2777 grafts registered by the UK Transplant Support Service from July 1987 to June 1991. RESULTS Several recipient factors influencing graft survival, rejection, and visual acuity were identified, but no donor factors. Of the operative factors amenable to change, mixed suturing was associated with reduced graft survival, and larger grafts with increased risk of rejection but better visual acuity when surviving. There was increased risk of rejection with poor matching at HLA class I antigens, but mismatched HLA-DR grafts suffered less rejection than those with zero HLA-DR mismatches. Recipient age below 10 years was associated with increased risk of both rejection and graft failure. However, whereas increasing age above 10 years was not associated with differential graft survival, it was significantly associated with decreasing risk of rejection. CONCLUSIONS While confirming possible benefits of HLA-A and B matching, the expense and delay involved in awaiting matched HLA-DR tissue is unlikely to be justified. Other donor factors are unrelated to graft outcome following screening of tissue by eye banks. The highest rates of graft failure and rejection happen in the early postoperative period, and factors influencing visual outcome are also apparent at this stage.

Immune cell infiltration and persistence in the mouse trigeminal ganglion after infection of the cornea with herpes simplex virus type 1

Following inoculation of the mouse cornea with herpes simplex virus type 1 (HSV-1), the spread of virus was investigated and the types of immune cell infiltrating the trigeminal ganglion (TG) were identified in low temperature paraffin wax sections. Virus antigen was first found on day 3 and was absent after day 14. Early presentation of antigen to T cells may occur since increased expression of major histocompatibility complex (MHC) class II antigens, including de novo expression on satellite and Schwann cells, was detected in foci of such antigen on day 3. A second large peak of such expression was detected on day 10 together with increasing numbers of B and T cells. Large numbers of these lymphocytes and extensive expression of MHC class II were seen in the TG well into the phase of virus latency; the significance of this is discussed.

Changes in the collagenous matrix of the aging human lamina cribrosa.

AIMS--The age-related changes in the biochemical composition of the collagenous matrix of the human lamina cribrosa were investigated. METHODS--An age range (3 weeks to 92 years old) of human laminae cribrosae, dissected free of any surrounding structures which contained collagen, were analysed for collagen solubility (n = 58) total collagen content (n = 46), proportion of collagen types (n = 38), and collagen cross linking (n = 30), using hydroxyproline analysis, scanning densitometry of peptides after cyanogen bromide digestion, and high performance liquid chromatography, respectively. RESULTS--Age-related changes included an increase in total collagen and a decrease in the proportion of type III collagen within the lamina cribrosa. The collagen cross link pyridinoline was present at low levels, but demonstrated no trend with age. An age-related increase was found in pentosidine, an advanced glycation product. CONCLUSION--These changes in collagen composition imply that the mechanical properties of the lamina cribrosa are altered, resulting in a stiffer, less resilient structure with age. Such alterations in structure may contribute to the increased susceptibility of the elderly to axonal damage in chronic open angle glaucoma.

Preparation of Bruch's membrane and analysis of the age-related changes in the structural collagens.

AIMS/BACKGROUND--The morphological changes in Bruchs membrane and its constituent collagen seen during aging have been studied extensively but the chemical nature of the collagen and any aging changes have not previously been evaluated. METHODS--A method for preparing purified Bruchs membrane from human cadaver eyes by dissection preceded by trypsin digestion was developed. Following pepsin digestion, the constituent collagens were analysed by SDS-PAGE and by immunoblotting. Cyanogen bromide digestion was used to ascertain the solubility of the collagen and the proportion of type I to type III collagen. After hydrolysis of Bruchs membrane samples the constituent amino acids and collagen crosslinks were measured. RESULTS--The presence of collagen types I, III, IV, and V in Bruchs membrane was confirmed. The proportion of type III collagen as a percentage of total fibrous collagens was calculated as being between 35% and 39%, with no significant difference between different macular and peripheral sites or with age. There was a highly significant decline in the solubility of Bruchs membrane collagen with age, from near 100% in the first decade of life to 40-50% in the ninth decade at both macular and peripheral sites. There was no significant change in the amount of enzymatically formed collagen crosslinks with age. Amino acid analysis indicated a significant increase in the amount of non-collagen protein with age in macular but not peripheral sites. CONCLUSION--Changes in the constituent collagens may contribute to the accumulation of debris in Bruchs membrane with age and interfere with the function of the retinal pigment epithelium, with subsequent consequences for the overlying photoreceptors.

Cytokine production in the nervous system of mice during acute and latent infection with herpes simplex virus type 1.

Immunocytochemistry on serial paraffin sections was used to monitor the production dynamics of cytokines (IL-2, IL-4, IL-6, IL-10, IFN-gamma and TNF-alpha) and viral antigens in the trigeminal ganglion (TG) and the central side of the dorsal root entry zone (DRE) of mice, following infection of the cornea with herpes simplex virus type 1. In normal TG, scattered satellite cells were TNF-alpha+ and in the DRE, TNF-alpha+ and/or low numbers of IL-6+ cells were detected. On day 3 after infection, foci of TG neurons with viral antigens were surrounded by large numbers of TNF-alpha+ and/or IL-6+ cells and low numbers of IFN-gamma+ cells. IL-2+ and/or IL-4+ cells appeared later, when viral antigens had almost cleared. In the TG, the most striking changes occurred with TNF-alpha, with respect to its source (satellite cells, Schwann cells and infiltrating cells) and the extent and long duration of its production. TNF-alpha was the predominant cytokine throughout acute and latent infection and even by day 30, numbers of satellite cells expressing this cytokine were three times higher than those in normal ganglia. Moreover, in the DRE, TNF-alpha was the only cytokine detected during virus clearance and again, its production continued, along with that of IL-6, on days 20 to 30, in both infiltrating cells and astrocytes. Thus, cytokines, particularly TNF-alpha and perhaps IL-6, from infiltrating cells and resident glial cells may have a role both in virus clearance and in normal homeostatic mechanisms in the nervous system such as repair and protection of neurons from damage.

Reactivation of latent infection and induction of recurrent herpetic eye disease in mice.

During primary ocular infection of mice with herpes simplex virus type 1 (HSV-1) strain McKrae, dendritic corneal ulcers developed and many eyes became permanently damaged. When primary infection had subsided, latent infection was detected in the three parts of the trigeminal ganglion and in the superior cervical ganglion. Such latently infected mice were treated with cyclophosphamide, dexamethasone and u.v. irradiation, or cyclophosphamide and dexamethasone alone. After treatment with immunosuppressive drugs and u.v. irradiation infectious virus was isolated from the ophthalmic part of the trigeminal ganglion, and in eyelids and eyewashings; recurrent herpetic eye disease was seen but only in eyes undamaged by primary infection. After treatment with cyclophosphamide and dexamethasone alone there was a lower incidence of virus isolated from eyewashings and no recurrent disease was seen. There was a good correlation between the pattern and distribution of recurrent lesions and the distribution of cells stained due to the presence of virus antigens.

Acanthamoeba keratitis: risk factors and outcome.

AIMS/BACKGROUND--This study was initiated to investigate risk factors for and outcome of Acanthamoeba keratitis. METHODS--Results of treatment were studied in 22 patients (23 eyes) presenting to Bristol Eye Hospital between 1985 and February 1995. Details related to the use and disinfection of contact lenses were also obtained. An additional two patients who were seen at Bristol but mainly treated elsewhere were surveyed for contact lens related information only. RESULTS--The incidence of Acanthamoeba keratitis rose substantially in the 1990s: three patients presented before 1990, while the remaining 21 presented between January 1990 and February 1995. Eleven patients have presented since january 1994. All of the patients in this series were contact lens wearers, 16 (67%) using daily wear disposable contact lenses. Contact lens disinfection data were available in 22 patients of whom 11 (50%) were using chlorine disinfectant. Other types of disinfection were much less common. Four patients (18%) had not used any disinfectant. During the course of the series the average diagnostic delay has fallen markedly, although in 77% of patients a diagnosis of a viral keratitis, most commonly herpes simplex, was made on first presentation. All but three of the series were treated with a combination of polyhexamethylene biguanide and propamidine isethionate. Penetrating keratoplasty was performed in 9/23 eyes (39%); in all of these eyes diagnosis was delayed for at least 6 weeks. All but one of the eyes in the series achieved a visual acuity of 6/9 or better after treatment, and 18 eyes (78%) saw 6/6 or better. CONCLUSIONS--Most patients with Acanthamoeba keratitis can now expect a good visual result and cure by medical therapy alone is favoured by early diagnosis.

Human herpesviruses in the cornea.

AIMS To determine the sensitivity and specificity of culture, immunohistochemistry (IHC), the polymerase chain reaction (PCR), and in situ hybridisation (ISH) for detecting herpes simplex virus (HSV-1) in the cornea of patients undergoing penetrating keratoplasty. To compare the incidence of HSV-1 in the cornea with that of varicella zoster virus (VZV), cytomegalovirus (CMV), and Epstein-Barr virus (EBV). METHODS The corneas of 110 patients, 52 with a documented history of herpes keratitis (HSK) and 58 with non-herpetic corneal disease, were investigated using IHC, PCR, ISH, and culture. RESULTS HSV-1 DNA and antigen were detected in 82% and 74% respectively, of corneas of patients with HSK and in 22% and 15% of corneas of patients with no history of HSK. The sensitivity of PCR and IHC was 82% and 74% with a specificity of 78% and 85%, respectively. HSV-1 DNA and antigen were found more frequently and in increased amounts in corneas of patients with a short interval between their last attack of HSK and surgery. There was a good correlation between PCR and IHC in 71%. HSV-1 was isolated by culture in 2%. Latency associated transcripts were not detected using ISH. Evidence of VZV DNA or antigen was found significantly more frequently in the corneas of patients with a history of HSK (p<0.001). No evidence of EBV or CMV was found in any cornea. CONCLUSIONS PCR and IHC are both sensitive for the detection of HSV-1 in the cornea. A combination of PCR and IHC increases the specificity for the diagnosis of HSK to 97%. HSV-1 appears to be slowly removed from the cornea. VZV and HSV-1 may co-infect the cornea.

Spread of virus and distribution of latent infection following ocular herpes simplex in the non-immune and immune mouse.

In both non-immune and immune mice infected with herpes simplex virus the incidence of latent infection of the trigeminal ganglion was related to the severity of ocular virus infection. During primary infection, virus was shown to travel via the ophthalmic part of the ganglion to reach the brainstem, from where centrifugal spread resulted in latent infection of neurones in the trigeminal ganglion which did not serve the site of inoculation. Primary infection also resulted in latent infection of the superior cervical ganglion. Shedding of virus occurred rarely in the tears of animals which had recovered from primary disease. In immune mice, spread of virus resulted in a much lower incidence of latent infection and that occurred only in ophthalmic neurones.