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A modified tandem scanning confocal microscope was used for real-time in vivo examination of the rabbit cornea following a cryogenic injury. The corneas of New Zealand white rabbits were frozen with a probe that had been cooled by immersion in liquid nitrogen, effectively destroying keratocytes in a central 5 mm diameter zone throughout the total thickness of the cornea. In these eyes, keratocyte repopulation and corneal stromal wound healing proceeded similarly to that which occurs after epikeratophakia, a refractive surgical procedure designed to change the curvature and optical power of the cornea. In epikeratophakia, a cryolathed donor corneal stroma lenticule is sutured onto the bare stroma of the recipient cornea. The collagen tissue lenticule is repopulated by keratocytes (corneal fibroblasts) that migrate in from the host cornea. In our study, the confocal microscope permitted sequential, noninvasive examination of the corneal stroma in the treated animals. Necrosis of the keratocytes, followed by activation of the remaining viable cells in the corneal periphery, was observed in the first 2 to 3 days after cryo injury. A fine stromal fibrous network was seen to develop; in three eyes, this network progressed to the development of a retrocorneal fibrous membrane and dense stromal fibrosis, both of which resulted in significant loss of corneal clarity. Our results suggest that the confocal microscope may be a valuable tool to provide much needed information on wound healing processes at the cellular level after corneal surgery and injury.

Five patients with Down's syndrome underwent penetrating keratoplasty for keratoconus. In three patients, the indication for surgery was acute corneal hydrops, which had not resolved in the three months before surgery. The other two patients had corneal scars. Two patients had combined penetrating keratoplasty, cataract extraction, and intraocular lens insertion. Four of the five patients maintained clear grafts at their most recent follow-up examination. Two of the five patients had one or more graft reaction episodes; one graft was lost. Good results can be obtained in penetrating keratoplasty for keratoconus in patients with Down's syndrome who do not demonstrate a tendency toward excessive eye rubbing and for whom a single observant caretaker can be relied on to provide consistent postoperative care.

An understanding of the cellular genes whose expression is altered during HSV reactivation will enable us to better understand host responses and biochemical pathways involved in the process. Furthermore, this knowledge could allow us to develop gene-targeted inhibitors to prevent viral reactivation. Mice latent with HSV-1 strain McKrae and uninfected control mice were subjected to hyperthermic stress (43 degrees C for 10 min) and their trigeminal ganglia (TG) collected 1 h later. Two additional groups included HSV-1 latently infected and uninfected mice not subjected to hyperthermic stress. Poly A+ mRNA was enriched from total mouse TG RNA and reverse transcribed using MMLV RT. Radioactively labeled cDNAs were analyzed by microarray analysis. A stress/toxicology array of 149 mouse genes on a nylon membrane was used. The labeled cDNAs prepared from latently infected, stressed mice demonstrated 3-fold or greater increases in certain mRNA-early response genes (ERGs) compared to cDNAs from uninfected, stressed control mice. The ERG mRNAs that showed increases included two heat shock proteins (HSP60 and HSP40), a basic transcription factor (BTF T62), a DNA repair enzyme, two kinases [MAP kinase and a stress-induced protein kinase (SADK)], an oxidative stress-induced protein, a manganese superoxide dismutase precursor-2 (SOD-2), and cyclooxygenase 2 (COX-2). The gene expression in unstressed, infected TGs was similar to the gene expression in unstressed, uninfected controls. These results suggest that there is a significant difference in the ERG expression profile in latently infected TGs undergoing stress-induced reactivation compared to uninfected TGs.

Keratocytes express MHC class I molecules constitutively, and keratocytes stimulated with IFN-gamma express MHC class II molecules. Unstimulated keratocytes constitutively express B7-1 and ICAM-1, as well as low levels of CD40 and 4-1BBL. These findings indicate that keratocytes may deliver both antigen-specific and costimulatory signals to CD4(+) and CD8(+) T cells. To demonstrate that keratocytes expressing B7-1 provide a costimulatory signal to T cells, CD4(+) or CD8(+) mouse T cells were incubated with anti-CD3 mAb and irradiated keratocytes. Enhanced proliferation of both CD4(+) and CD8(+) T cells occurred, and could be inhibited by anti-B7-1 mAb, indicating T cell costimulatory activity by B7-1 on the keratocytes. To demonstrate that keratocytes can deliver an antigen-specific signal, CD4(+) and CD8(+) T cells from herpes-infected mice were incubated with HSV-1-infected, irradiated keratocytes. The resulting T cell proliferation and production of Th1 cytokines (IL-2, IFN-gamma) indicated T cell activation by antigens presented by the infected keratocytes. These results show that keratocytes in the corneal stroma of the mouse can function as antigen-presenting cells and, thus, may play a role in immune-mediated stromal inflammation such as herpetic stromal keratitis.

The2',5'-oligoadenylate-dependent RNase L gene functions in the interferon-inducible RNA decay pathway known as the 2-5A system. The purpose of this study was to determine whether the absence of this gene affects the pathogenesis of herpes simplex virus type 1 (HSV-1) ocular infection in the mouse.HSV-1 (strain McKrae) was applied bilaterally to unscarified corneas of RNase L-null mice and congenic controls. To evaluate the severity of herpetic keratitis, slit lamp examinations (SLE) were performed every other day for 14 days. To study corneal histology and apoptosis, HSV-1-inoculated RNase-L-null and congenic control mice, as well as mock-inoculated mice (apoptosis negative control), were killed at 6 and 18 hours postinoculation (PI). Uninoculated mice that underwent corneal scarification (apoptosis positive control) were killed 2 hours after scarification. Eyes were dissected and the corneas processed for light and transmission electron microscopy and the TUNEL assay.In comparison with the congenic control mice, RNase L-null mice showed significantly more severe herpetic keratitis (PI day 8, SLE score, mean +/- SEM: 3.27 +/- 0.10 vs. 2.34 +/- 0.06; P:0.001) and significantly higher mortality (PI day 14, 70% vs. 20%; P:0.001). Few apoptotic cells were seen in HSV-1-infected RNase L-null mice, although DNA fragmentation consistent with apoptosis was detected in the corneas of congenic control mice 6 and 18 hours after HSV-1 inoculation and in uninfected mice with scarified corneas. Signs of apoptosis were not present in the mock-infected corneas. Electron microscopic evidence of keratocytic apoptosis was detected only in the uninfected scarified corneas and the HSV-1-infected congenic control corneas.The increased severity of ocular disease and increased mortality in the RNase L-null mice provides evidence, for the first time, that the 2-5A system contributes to protection during ocular herpetic infection. The reduced frequency of apoptosis in these mice suggests that one possible mechanism for this protective effect could be the induction of apoptosis in corneal cells as a means of reducing the spread of infectious virus.

Ocular virus infections remain an important cause of corneal and external disease. Herpes simplex, the most important, is easily treated when it is confined to the epithelium. New studies indicate that herpetic stromal disease and iritis are effectively treated with a combination of corticosteroid and antiviral without additional risk. Recurrences of ocular herpetic disease can be reduced with acyclovir given orally; the benefit seems to be greatest in patients who have had at least one episode of stromal keratitis. Herpes zoster can be treated with either acyclovir or famciclovir, but to be effective, treatment must be initiated within 72 hours of onset. Early treatment reduces the risk of post-herpetic neuralgia and may reduce the risk of ocular complications. Adenovirus infection (epidemic keratoconjunctivitis) is often spread by the ophthalmologist. New medications such as cidofovir appear to be effective against the adenoviruses in non-human systems and may have some effect in man, although previously, drugs that appeared to have an effect in vitro have proven to be ineffective in the clinical setting.

To investigate the migration of herpes simplex virus type 1 (HSV-1) between latently infected and naive corneal tissues and trigeminal ganglion (TG) in rabbits after penetrating keratoplasty (PKP) and transcorneal epinephrine iontophoresis.Two mutants, genetically constructed from HSV-1 strain 17syn+, were used to inoculate rabbit corneas: 17deltaPst, a latency associated transcript (LAT) negative, low-reactivating virus and 17Pr, a high-reactivating, LAT-positive rescuant of 17deltaPst. Latently infected rabbits were given corneal allografts from naive rabbits, and naive rabbits received grafts from latently infected rabbits. Ninety days after PKP, groups of the transplanted rabbits were induced to reactivate by transcorneal epinephrine iontophoresis, but others were not induced. Viral shedding was monitored by tear film cultures. Rabbits were killed 5 days after iontophoresis. Transplanted grafts, recipient corneal rims, and corresponding TG were obtained. Nucleic acids were extracted and amplified for detection of HSV-1 DNA and viral gene transcription.In naive rabbits receiving grafts transplanted from rabbits latently infected with 17Pr (LAT+), 3 of 6 corneal rims contained HSV DNA after induction. In contrast, none of the 5 corneal rims from naive rabbits receiving grafts from rabbits latent with 17deltaPst (LAT-) contained viral DNA. Viral DNA and gene transcripts were detected in 2 of 6 TG from naive rabbits that received grafts from 17Pr (LAT+) latently infected rabbits. In recipient corneal rims and TG of latently infected rabbits receiving grafts from naive rabbits, viral DNA concentration was significantly greater with induced reactivation, compared with the results in noninduced rabbits. The amount of viral DNA in naive grafts transplanted into 17Pr (LAT+) latently infected rabbits was significantly higher with induction than without induction (P = 0.018). More viral DNA and viral gene transcripts were found in tissues from rabbits latently infected with 17Pr (LAT+) than in rabbits latently infected with 17deltaPst (LAT-).Corneas from latently infected rabbits contain HSV-1 DNA that can replicate after induced reactivation. Viral migration can occur in both anterograde and retrograde directions between the transplanted graft and the recipient corneal rim and TG. The LAT negative HSV-1 construct 17deltaPst has a significantly reduced ability to replicate and migrate.

In this study, we determined the binding characteristics of F(ab')2 alloantibody fragments to corneal antigens and assessed the capacity of these antibody fragments to protect corneal allografts from immune attack.Goat anti-rabbit alloantibodies were pepsin-digested and labeled with 125I, and the time course of association and dissociation of the F(ab')2 fragments was determined. Corneal allografts were incubated in unlabeled F(ab')2 fragments and transplanted into allogeneic recipients, and the graft survival times were recorded.Binding of radiolabeled F(ab')2 fragments to rabbit cornea cells reached a maximum at 12 hr. At 32 degrees C (rabbit corneal temperature), the radiolabel eluted rapidly from the cornea, reaching baseline at 72 hr. At 4 degrees C (corneal graft storage temperature), significant amounts remained associated with the cornea at 96 hr. Mean survival time for grafts incubated in F(ab')2 anti-rabbit fragments was significantly greater than that of grafts incubated in nonimmune F(ab')2 fragments. Three of the corneal allografts incubated in goat F(ab')2 anti-rabbit fragments survived for 100 days, whereas the longest surviving control allograft incubated in goat F(ab')2 nonimmune fragments was rejected on day 24. Preincubation of corneas in unlabeled, immune F(ab')2 fragments followed by incubation in radiolabeled, immune F(ab')2 fragments suggested that antigen masking was not a factor in the prolongation of graft survival.Based on the binding and release kinetics and the graft survival times, it appears that the protective effect of immune F(ab')2 fragments extends well beyond the binding interval of the antibody fragments to corneal cell membranes.

This study aimed to detect corneal conditions presenting with linear images on white light confocal microscopy and to analyze their distinguishing characteristics.In 1996 and 1997, 153 eyes of 110 patients with various corneal conditions were examined. In vivo examination of the cornea was performed by using a white-light tandem scanning confocal microscope. Images were captured by using a video camera and stored on S-VHS video tapes. In this retrospective study, patient charts and confocal microscopic video records were reviewed. Conditions with linear images were looked for, and the images were analyzed and compared.The only structures presenting as linear images on confocal microscopy in normal subjects consisted of corneal nerves. The following pathologic conditions also had linear images on confocal microscopy: corneal vascularization, mycotic keratitis, lattice corneal dystrophy, and posterior polymorphous dystrophy. Each condition could be identified based on its reflectivity, delineation, size, branching pattern, and location in the cornea.Different corneal conditions present with linear images on confocal microscopy. Correct identification is critical to avoid misdiagnosis.

We used real-time scanning confocal microscopy to evaluate early changes in corneal wound healing after excimer laser photorefractive keratectomy (PRK). Adult New Zealand White rabbits were given photorefractive keratectomy treatments appropriate for 5.00 to 8.00 D of myopia (44.5 to 71.0 micros depth, with a 5-mm diameter treatment zone). Daily confocal microscopic examinations showed acute loss of keratocytes in the anterior corneal stroma by 5 hours; losses were maximal between 24 and 48 hours for 5.00 D and 6.00 D ablations and between 72 and 96 hours for 7.00 D and 8.00 D ablations. The oval nuclei of normal keratocytes gave way to spindle-shaped fibroblasts accompanied by an accumulation of fibrillary extracellular matrix. Fibroblasts density increased toward the end of the week. Deeper ablations resulted in a longer period of keratocyte depletion and delayed onset of fibroblast activity. No epithelial, deep stromal, or endothelial abnormalities were detected, nor was stromal inflammation found. Light microscopy 1 week after PRK revealed superficial fibroplasia, which correlated with the en face images obtained with real-time in vivo confocal microscopy. The confocal microscope has a number of advantages as a clinical tool for investigation of laser-induced changes in corneal keratocytes and the stromal matrix, which may play a role in determining visual outcome after PRK.

Corneal allografts were transplanted into inflamed and vascularized graft beds in rabbit eyes. The grafts were examined every 4 days by slit-lamp biomicroscopy and scanning confocal microscopy. Confocal images were recorded with a video camera and computer enhanced in real-time. Layers of the cornea were visualized in serial optical sections parallel to the epithelium. In the third postoperative week, signs of graft rejection were observed; slit-lamp examination revealed a circumferential line of epithelial rejection, along with cloudiness and edema. Vessels were observed growing into the graft. By confocal microscopy, infiltrating cells were seen in the graft stroma. Foci of cells were especially pronounced around the sutures. Scattered leukocyte infiltrates were prominent at capillary terminals. There was an accompanying reduction in the stromal keratocyte density in the region of the infiltrate. Additionally, various degrees of fibrosis were noted around each suture and at the host-graft interface. Confocal microscopy may provide a valuable clinical tool for determining the earliest indicators of an antigraft immune response, and as an aid in the differential diagnosis of other inflammatory conditions of the cornea.

Models of recurrent herpetic keratitis that depend on tissue damage or immunosuppression have been described. The authors report a model that depends only on minimal temperature stress to produce clinical recurrences in a small primate.Squirrel monkeys (Saimiri sciureus) infected by the ocular route with the Rodanus strain of herpesvirus type 1 (HSV-1) were exposed to temperatures approximately 5 degrees C lower than the usual ambient temperature for periods as short as 12 hours.The corneas showed more or larger lesions typical of recurrent herpetic keratitis than are usually seen in these animals under normal conditions. Statistical analysis showed that there were significantly higher frequencies of epithelial keratitis at 18 degrees C and 20 degrees C (P0.0001).A minimal temperature change produced significant recurrences in this small animal within a short time. Tissues were not damaged to produce the recurrences. This approach may provide an efficient primate model for rapid testing of drugs to prevent clinical recurrence of ocular herpetic keratitis.

To evaluate the efficacy of topical corticosteroids in treating herpes simplex stromal keratitis.The authors performed a randomized, double-masked, placebo-controlled, multicenter clinical trial of 106 patients with active herpes simplex stromal keratitis who had not received any corticosteroids for at least 10 days before study enrollment. Patients were assigned to the placebo group (n = 49) or the steroid group (topical prednisolone phosphate; n = 57); both regimens were tapered over 10 weeks. Both groups received topical trifluridine. Visual acuity assessment and slit-lamp biomicroscopy were performed weekly for 10 weeks, every other week for an additional 6 weeks or until removal from the trial, and at 6 months after randomization.The time to treatment failure (defined by specific criteria as persistent or progressive stromal keratouveitis or an adverse event) was significantly longer in the steroid group compared with the placebo group. Compared with placebo, corticosteroid therapy reduced the risk of persistent or progressive stromal keratouveitis by 68%. The time from randomization to resolution of stromal keratitis and uveitis was significantly shorter in the steroid group compared with the placebo group even though both groups included patients who were removed from the study and treated with topical corticosteroids according to best medical judgment. Nineteen (33%) of the steroid-treated patients and 11 (22%) of the placebo-treated patients completed the 10 weeks of protocol therapy and had stable, noninflamed corneas after 16 weeks. At 6 months after randomization, no clinically or statistically significant differences in visual outcome or recurrent herpetic eye disease were identified between the steroid and placebo groups.The topical corticosteroid regimen used in this study was significantly better than placebo in reducing persistence or progression of stromal inflammation and in shortening the duration of herpes simplex stromal keratitis. Postponing steroids during careful observation for a few weeks delayed resolution of stromal keratitis but had no detrimental effect as assessed by visual outcome at 6 months.

To evaluate the efficacy of oral acyclovir in treating stromal keratitis caused by herpes simplex virus (HSV) in patients receiving concomitant topical corticosteroids and trifluridine.The authors performed a randomized, double-masked, placebo-controlled, multicenter trial in 104 patients with HSV stromal keratitis without accompanying HSV epithelial keratitis. Sample size was chosen so that a 5%, one-tailed test would have an 80% chance of detecting a doubling of the median time to treatment failure. Patients were randomized to receive a 10-week course of either oral acyclovir (400 mg 5 times daily, n = 51) or placebo (n = 53). All patients also received a standard regimen of topical prednisolone phosphate and trifluridine. Ophthalmologic examinations were performed weekly during the 10-week treatment period, every 2 weeks for an additional 6 weeks, and at 6 months after entry into the trial.The median time to treatment failure (defined as worsening or no improvement of stromal keratitis or an adverse event) was 84 days (95% confidence interval, 69-93 days) for the acyclovir group and 62 days (95% confidence interval, 57-90 days) for the placebo group. By 16 weeks, 38 patients (75%) in the acyclovir group and 39 patients (74%) in the placebo group had failed treatment. Also by that time, the keratitis had resolved with trial medications, and there was no subsequent worsening in nine patients (18%) in the acyclovir group and ten (19%) in the placebo group. None of these results were significantly different between the two groups. However, visual acuity improved over 6 months in significantly more patients in the acyclovir group than in the placebo group.There was no statistically or clinically significant beneficial effect of oral acyclovir in treating HSV stromal keratitis in patients receiving concomitant topical corticosteroids and trifluridine with regard to time to treatment failure, proportion of patients who failed treatment, proportion of patients whose keratitis resolved, time to resolution, or 6-month best-corrected visual acuity. Visual acuity improved over 6 months in more patients in the acyclovir group than in the placebo group.

A modified tandem scanning confocal microscope was used for real-time in vivo examination of the rabbit cornea following a cryogenic injury. The corneas of New Zealand white rabbits were frozen with a probe that had been cooled by immersion in liquid nitrogen, effectively destroying keratocytes in a central 5 mm diameter zone throughout the total thickness of the cornea. In these eyes, keratocyte repopulation and corneal stromal wound healing proceeded similarly to that which occurs after epikeratophakia, a refractive surgical procedure designed to change the curvature and optical power of the cornea. In epikeratophakia, a cryolathed donor corneal stroma lenticule is sutured onto the bare stroma of the recipient cornea. The collagen tissue lenticule is repopulated by keratocytes (corneal fibroblasts) that migrate in from the host cornea. In our study, the confocal microscope permitted sequential, noninvasive examination of the corneal stroma in the treated animals. Necrosis of the keratocytes, followed by activation of the remaining viable cells in the corneal periphery, was observed in the first 2 to 3 days after cryo injury. A fine stromal fibrous network was seen to develop; in three eyes, this network progressed to the development of a retrocorneal fibrous membrane and dense stromal fibrosis, both of which resulted in significant loss of corneal clarity. Our results suggest that the confocal microscope may be a valuable tool to provide much needed information on wound healing processes at the cellular level after corneal surgery and injury.

We firstly report an experimental study on the sensitivity of primary cultural human trigeminal ganglionic cells infected by herpes simplex virus type 1 (HSV-1) in different time-points. The results indicate that the neuron of human trigeminal ganglia is the most sensitive cell type, the antigen of HSV-1 replicates rapidly within the cytoplasm and cell nucleus, the fibroblasts and glia cells are infected partly by the virus. Therefore, perhaps the neuron is the main harboring cell type.

The collagen shield is a contact lens-shaped collagen matrix that can be impregnated with a drug during hydration or manufacture and then, when placed on the cornea, acts as a reservoir, dissolving slowly and releasing the drug over time. This new ocular drug delivery system has proven useful in the treatment of corneal infections and the prevention of graft rejection. The lubricating effect of the dissolving shield matrix may palliate dry eye symptoms, but the shields are not useful as a treatment for keratoconjunctivitis sicca because they must be applied in the physician's office and they are not transparent. A new formulation of collagen and cetyl alcohol particles suspended in a methylcellulose vehicle (Lacrisomes) is being developed to relieve symptoms in dry eye patients. A similar system, with the addition of therapeutic agents during manufacture (Collasomes), is being tested for drug delivery to the ocular surface. The advantages of both of these systems would be their ease of application and minimal interference with vision.

The tandem scanning confocal microscope (TSM) was adapted for in vivo examination of the cornea in rabbits with experimental bacterial and fungal keratitis. Compared to slit lamp biomicroscopy, the TSM provides superior lateral and axial resolution and serial optical sectioning capability, which may be useful for identification of corneal pathogens in the early stages of infection. We used the TSM to examine normal rabbit eyes infected with bacteria (Bacillus cereus) and a filamentous fungus (Aspergillus). We also examined a human cornea removed by penetrating keratoplasty after a clinical diagnosis of amoebic keratitis. In the early stages of bacterial infection, slit lamp examination revealed a nonspecific minimal stromal haze and limbal injection indistinguishable from sterile ulcers and epithelial defects. With the TSM, bacteria were visible as highly refractile bodies in the epithelium and superficial stroma. Branching fungal hyphae were also easily identified by the TSM, as were Acanthamoeba cysts and parasites in the subepithelial stroma. Our results indicate that this technique may provide a new modality for quickly and accurately identifying the agent of corneal infection, thereby facilitating prompt and appropriate treatment.

Ten partially sighted and 19 normally sighted eyes underwent excimer laser photorefractive keratectomy for the correction of myopia. Nine of the partially sighted and 17 of the normally sighted eyes had 12 months of follow-up. Epithelial healing was complete in all eyes by day 6. None of the eyes had recurrent erosions, infections, or other medical complications. An increase in corneal haze after surgery was followed by a slow trend toward clearing. Average uncorrected visual acuity in the 7 normally sighted eyes with attempted corrections of 5 diopters (D) or less was 20/40 from month 2 on; the eyes with greater than 5 D attempted corrections had an average of 20/80--at month 2, which declined to 20/200--by month 6. Best spectacle-corrected visual acuity was within +/- 1 Snellen line of preoperative values in 14 of the normally sighted eyes, improved 2 or more lines in 2 eyes, and worsened two or more lines in two eyes. Hard contact lens overcorrection restored all of the two-line loss in 1 eye and 1 line of the 3-line loss in the other. Refraction and keratometry indicated corneal flattening without induced astigmatism.

The authors first report in China the detection of HSV-1 antigen in 36 human corneas with recurrent HSK, using the horse radish peroxidase staining technique. The results were that in 16 eyes of quiescent HSK, the HSV antigen was negative, and in 11 of 20 corneas with active HSK, the HSV antigen was positive. When 5 corneas with quiescent HSK were cultured in vitro, 3 again became HSV antigen positive. The findings may be useful in the treatment of HSK.

Penetrating keratoplasty (PKP) in conjunction with postoperative corticosteroids may reactivate latent herpes simplex virus type 1 (HSV-1) to cause persistent postoperative epithelial defects. The clinical diagnosis of HSV keratitis after penetrating keratoplasty is difficult because the postoperative appearance may be nondendritic and, therefore, not characteristic of HSV-1 infection. Presently, the most reliable method to diagnose HSV-1 under these conditions is to culture eyes for the presence of HSV-1. To determine the coincidence of positive HSV-1 ocular cultures with HSV-1 epithelial defects, 15 rabbits (20 eyes) latently infected with HSV-1 underwent autograft PKP with postoperative corticosteroids. Daily ocular cultures and slit-lamp examinations were performed on postoperative days 1-8 and 10. Viral shedding occurred in 15 of 19 (79.0%) of the eyes postoperatively. Superficial punctate keratopathy (SPK) was observed in 19 of 19 (100%) of the eyes and coincided with positive HSV-1 cultures 24% of the time. Dendritic lesions were observed in three of 19 (15.8%) of the eyes; the dendrites coincided with positive HSV-1 cultures 60% of the time. Finally, epithelial ulcers were seen in eleven of 19 (57.9%) of the eyes, thus coinciding with HSV-1 positive cultures 29% of the time. The greatest coincidence of positive HSV-1 cultures with nondendritic epithelial lesions occurred on postoperative day number 4. The results suggest that an epithelial lesion following PKP and postoperative corticosteroids could represent HSV infection, even if a single HSV ocular culture is negative.

Collagen shields were tested as a means of delivering the immunosuppressive drug, cyclosporine A (CsA), to the cornea and aqueous humor in rabbit eyes. Gelatinous collagen was mixed with crystalline CsA and then, during drying, was formed into contact lens-shaped shields which were applied to rabbit eyes. The amount of CsA in the corneas and aqueous humor (AH) samples from shield-treated eyes was compared with samples from rabbit eyes treated with CsA in olive oil. CsA concentrations were measured by radioimmunoassay (RIA) at 2, 4, and 8 h after application of the shields or drops. Both the corneal and aqueous humor concentrations of CsA achieved with the shield delivery system were 10-fold higher than those obtained with topical CsA-olive oil drops. The CsA levels achieved in the cornea using the collagen shield are sufficient to inhibit cellular immune reactions in vivo. These results demonstrate that collagen shields may be useful as an ocular delivery system for the drug CsA.

The authors compared postkeratoplasty astigmatism over a 4-month period after surgery in a randomized, prospective study of two groups of patients (total N = 18) who received two different suture techniques. The test group N = 8) had a single running suture with postoperative suture adjustment; on the basis of computer-assisted topographic analysis, the suture was tightened in the flatter meridian and loosened in the steeper meridian in the first month after surgery. The control group (N = 10) had a standard double running suture procedure with no postoperative adjustment; the single running 10-0 nylon suture was removed 3 months after surgery. Four months after penetrating keratoplasty, mean (+/- standard deviation) astigmatism in the test group was 1.7 +/- 0.7 diopters (D), and all patients had less than 2.6 D of astigmatism. In the control group, mean astigmatism was significantly higher (5.4 +/- 2.4 D; range, 0.7-9.0 D; P less than 0.01). The results suggest that postkeratoplasty astigmatism can be reduced with the single running suture technique accompanied by postoperative suture adjustment.

Autogenous periosteum was used to reinforce scleral thinning, perforation, or corneoscleral wound dehiscence in four eyes of three patients with necrotizing scleritis or peripheral ulcerative keratitis associated with advanced rheumatoid arthritis. All grafts have remained intact during an average follow-up interval of 36 months (range, 19-52 months). The postoperative visual acuity ranged between 20/30-20/60. Two eyes exhibited age-related macular degeneration, and one eye had an epiretinal membrane postoperatively. No systemic complications occurred following surgery.

Twelve eyes undergoing penetrating keratoplasty with a double-running suture technique had corneal topographical analysis immediately before and one month after 10-0 suture removal. Substantial changes in astigmatism were evident following suture removal. Twenty five percent of eyes showed a decrease of 1 D, 33% of eyes showed a decrease of 3 D, 8.3% of eyes showed an increase of 1 D, 8.3% of eyes showed an increase of 2 D, 16.6% of eyes showed an increase of 3 D, and 8.3% of eyes showed an increase of 4 D of astigmatism following suture removal. The mean corneal astigmatism did not show a significant change from a mean of 5.3 D after suture removal. There was less individual variation of spherical corneal power following suture removal. Thirty three percent of eyes showed a decrease of 1 D, 33% of eyes showed a decrease of 2 D, 8.3% of eyes showed a decrease of 3 D, 18.3% of eyes showed an increase of 1 D, and 6.6% of eyes showed an increase of 2 D of spherical power following suture removal. The surface asymmetry index (SAI), a centrally weighted measure of corneal surface irregularity, decreased significantly (P less than 0.04) from a mean of 1.17 before suture removal to a mean of 0.93 after suture removal. These results suggest that surface irregularity may be decreased following the removal of a single running 10-0 nylon suture following penetrating keratoplasty.

Over the past few years, our understanding of the absorption, distribution, and biotransformation of ophthalmic pharmacologic agents has increased markedly. Such advances, coupled with a greater appreciation of the ocular autonomic nervous system and of the dynamic and physical properties of biological and synthetic membranes, have resulted in the development of new drugs and drug delivery systems. In this chapter, we examine recent advances in glaucoma therapy, includ­ ing membrane drug-delivery systems; timolol, a ,a-adrenergic blocking agent; and a pharmacologic approach to reducing aqueous outflow resis­ tance by medical trabeculocanalotomy. In addition, we briefly review the current state of ocular antiviral therapy and the development of artificial tear inserts for the treatment of dry eyes.

The pattern of ocular disease produced in the rabbit eye by HSV-1 (F) and HSV-1(MP) strains and recombinants F(MP)A, F(MP)B, F(MP)C, F(MP)D, F(MP)E, and F(MP)F was studied. The characteristics of ocular herpetic disease such as morphology of dendritic ulcers, severity of epithelial disease and incidence and duration of stromal disease produced in the rabbit eye are genetically determined by the virus strain. Our studies show that transfer of a defined part of the genome of the stromal disease-producing virus, HSV-1(MP), to the genome of an epithelial disease-producing virus, HSV-1(F), yielded recombinants with one or more of the disease characteristics of the donor strain. Specifically, recombinant F(MP)D produced lesions characteristic of the donor HSV-1(MP) strain; recombinants F(MP)C and F(MP)E produced stromal disease approaching the severity of the disease produced by the donor HSV-1(MP) strain, and only recombinants F(MP)A and F(MP)B retained the typically elongate lesions of the recipient HSV-1(F), whereas the recombinant strain F(MP)F produced no disease. The viral functions pertaining to the ocular disease pattern map between 0.70 and 0.83 map units in HSV-1 DNA within the BglII F DNA fragment. The pattern of stromal disease is independent of the production of glycoprotein C and fusion of HEp-2-infected cells. The functions relating to these aspects of ocular disease segregate but are closely linked.

There are two antiviral agents that are commercially available for use in ocular herpes infections, idoxuridine, and vidarabine (Ara-A). In topical usage, these medications are similar in their effectiveness and toxicity; however, vidarabine is systemically active for treatment of deep ocular disease. Trifluorothymidine, which is still an experimental drug, has been shown in double-blind clinical trials to be the most potent antiviral drug for ocular use. These three antiviral drugs, however, are not truly selective in their action and interfere with normal cellular functions as well as virus synthesis. Several new drugs have been reported that are selective and are only effective in herpes virus infected cells; the most active of these compounds appears to be acycloguanosine.

Pyrimethamine (Daraprim) has been shown to kill proliferative Toxoplasmas in vivo and in vitro.1,2Although it is used for the treatment of toxoplasmosis and malaria in man, few observations on its pharmacology have been made in humans maintained on the drug. In antimicrobial therapy it is generally desirable to attain as rapidly as possible the highest concentration of a therapeutic agent consistent with acceptable levels of toxicity. The present study was undertaken (1) to determine an optimal therapeutic regimen for rapidly achieving high serum concentrations of pyrimethamine, (2) to elucidate the serum concentrations of the drug that are thereby achieved in man, and (3) to see whether a relation exists between the serum level of pyrimethamine and the bone marrow toxicity. Methods and Materials Nineteen patients being treated with pyrimethamine and sulfonamides were divided into three groups: 1. Multiple serum pyrimethamine assays were done on eight patients receiving 25

A series of keratoplasties was performed using 2 running nylon sutures of unequal size, 23 micrometer (10-0) and 16 micrometer. Visual acuities were assessed within 6 months of surgery and following removal of the 10-0 suture at 2 months. Thirty percent of aphakic and 80% of phakic had acuities of 20/70 or better.

We used specular microscopy to study the corneal endothelium of eight aphakic patients who had undergone epikeratophakia. The average post-operative period was 16 months with a range of 12 to 18 months. Corneal thickness increased from 0.52 mm +/- 0.01 mm to 0.88 mm +/- 0.04 mm with the addition of the onlay graft. The average pre-operative endothelial cell count was low (1,450 per mm2 +/- 218) and did not differ significantly (p greater than .9) from the average postoperative cell count (1,438 per mm2 +/- 218). Despite the increase in corneal thickness and low endothelial cell counts, we saw no corneal edema. We conclude that epikeratophakia is well tolerated by the cornea and can be performed safely on eyes that have already undergone substantial trauma to the endothelium.

Our clinical study was designed to determine whether "through-and-through" corneal sutures would prevent the postoperative intraocular pressure rise that occurs in 70% of aphakic eyes after uncomplicated penetrating keratoplasty. Twenty-five aphakic patients received the same operative procedure except that the corneal buttons of ten of them were sutured into place with "through-and-through" sutures. The intraocular pressures of patients with the "through-and-through" sutures were significantly lower than those of the other patients during the entire postoperative course.

The technique of using two continuous sutures of different caliber and relative tightness, running in the same direction, is described for penetrating keratoplasties. The primary purpose of the technique is to allow visual correction earlier in the postoperative period. In addition, graft slippage with override and posterior wound gape, both occurring at suture removal with resultant functional loss, have been eliminated.

The development of experimental disciform edema and necrotizing keratitis in the corneas or rabbits following intrastromal inoculation with the RE strain of herpes simplex virus is described. Following an initial episode of conjunctivitis and epithelial keratitis, a mild, centrally localized, stromal edema developed on the fifth day. Stromal edema, opcification, and neovascularization of the cornea reached maximum severity on the seventh to twenty-second day, and began to fade in most eyes thereafter. On the twenty-ninth day most corneas have attained a resolved state characterized by subepithelial granular opacities. Several eyes were observed which developed central necrotizing keratitis. Marked similarities between the animal model and human herpetic stromal keratitis were apparent. Histological observations show that early necrotizing keratitis in the rabbit is characterized by an infiltration of plasma cells and lymphocytes in the limbus, with polymorphonuclear leukocytes, lymphocytes, and macrophages in the central cornea.

Outflow facility was measured in 6 human eye bank eyes following penetrating keratoplasty and cataract extraction. A 7.5 mm corneal button was removed and replaced by an 8 mm button using conventional mid-to-deep stromal sutures. Baseline outflow facility in the phakic eyes (0.28 +/- 0.12 microliter/min/mm Hg, mean +/- SD) was unchanged (0.29 +/- 0.12) following the cataract extraction and penetrating keratoplasty using the larger corneal button.

Ten patients received retrobulbar bupivicaine (Marcaine) in conjunction with cyclocryotherapy for medically unresponsive postpenetrating keratoplasty glaucoma and were found to experience significantly longer postoperative pain than 20 patients who recieved lidocaine (Xylocaine) anesthesia. The duration of pain was correlated with the need for narcotic analgesis whenever possible. We encourage further investigation into the etiology of the discomfort after cyclocryotherapy employing retrobulbar bupivicaine anesthesia.