REFERENCES

1. Grizzard WS, Hilton GF, Hammer ME et al. Pneumaticretinopexy failures. Cause, prevention, timing, andmanagement. Ophthalmology 1995; 102: 929–36.

2. Wirostko WJ, Han DP, Perkins SL. Complications ofpneumatic retinopexy. Curr Opin Ophthalmol 2000; 11:195–200.

3. Taher RM, Haimovici R. Anterior chamber gas entrap-ment after phakic pneumatic retinopexy. Retina 2001;21: 681–2.

Upregulation of the productionof interferon-g and the geneexpression of the inducible nitricoxide synthase after excimer laserablation in rat corneas

The inflammatory response in the early stages of woundhealing after excimer laser phototherapeutic keratectomy(PTK) has previously been found to be mediated by infil-trating macrophages, as well as by corneal epithelial andendothelial cells.1

The formation and accumulation of free radicals, such asthose from nitric oxides (NO), also known as reactivenitrogen intermediates (RNI), has also been reported inbovine corneal strips after excimer laser irradiation.2 Wehave here analysed the expression of interferon-g (IFN-g)and that of NO/RNI in relation to the gene expression ofthe inducible NO-synthase (iNOS) 1, 12 and 24 h afterPTK treatment in rat corneas.

Nine eyes of nine Wistar rats received excimer laserPTKs (Chiron laser, 68 mm/pulse, diameter 4–7.6 mm,1606 pulses, depth in cornea 50 mm (Chiron TechnolasGmbH, Dornach, Germany)), and the rats (three groups of3 rats each) were sacrificed 1, 12 and 24 h after treatment.An additional group of three rats without PTK treatmentserved as control group. From all the collected samples,4–5 mm paraffin sections were obtained in RNAase-freeconditions. In situ mRNA hybridizations were carried outusing an iNOS-specific gene probe labelled with digoxi-genin, as previously described.3 Detection of hybrids wasperformed with the digoxigenin detection system fromRoche Diagnostics GmbH (Mannheim, Germany). Thescoring procedure was repeated at least three times bythree different evaluators. Plasma samples were collectedfrom treated and untreated animals shortly beforesacrificing. IFN-g was analysed in plasma samples byusing a commercially available EASIA/ELISA Kit (Bio-source Europe, Nivelles, Belgium). The concentration ofNO/RNI was evaluated as the end concentration of nitritein plasma by using a compensated Griess reaction,

Table 1. Gene expression of iNOS in corneal sections from Wistar rats, as detected by specific non-radioactive in situ hybridizations,†

compared with the concentrations of IFN-g (pg/mL) and NO/RNI (nmol/mL) in plasma, 1, 12 and 24 h after PTK

Time‡ after PTK (h) IFN-g (pg/mL)(means � SEM),

P (t, t - 1)

iNOS gene expression score(means � SEM), P (t, t - 1)

NO/RNI (nmol/mL)(means � SEM), P (t, t - 1)

0 (controls) (7.5 � 1.8) +/- (0.7 � 0.3) (16.1 � 4.9)1 (15.4 � 1.7)* P = 0.0298 + (1.7 � 0.2)* P = 0.0152 (41.7 � 3.8)** P = 0.007312 (22.9 � 2.3)*

P = 0.0491+++ (3.6 � 0.5)*

P = 0.0105(70.4 � 6.5)**

P = 0.009624 (25.1 � 1.2) P > 0.05 ++ (2.6 � 0.3) P > 0.05 (49.5 � 3.9)* P = 0.0258

PCC§ (to IFN-g) (1.0) 0.907 0.871PCC (to iNOS) 0.907 (1.0) 0.984PCC (to NO/RNI) 0.871 0.984 (1.0)

*The stars in the table indicate statistically significant values (1 star: P < 0.05; 2 stars: P < 0.01), when compared with the correspond-ing values observed for the time point immediately before (P [t, t - 1]). The P-values were calculated by the unpaired Student’s t-test.†Scores used for evaluation of the in situ hybridization results: (- = 0) negative reaction/not detected; (+/- = 1) positive reaction, <5%positive cells; (+ = 2) clearly positive reaction, approximately 5–10% positive cells; (++ = 3) extended positive reaction, 10–20% positivecells; (+++ = 4) very extended positive reaction, 20–50% or more positive cells. The evaluation procedure was repeated at least threetimes, by three independent evaluators, and was always performed without any sample reference. ‡Time indicates the time point in whichthe animals were sacrificed, in hours after the PTK treatment was completed. The presented values are means and standard error of themeans (SEM). §Pearson’s correlation coefficients (PCCs) were calculated between the analysed markers. PCCs are known to vary between1.0 (maximum) and 0.0 (minimum), so indicating the ‘maximum’ and ‘minimum’ levels of correlation, respectively. IFN-g, interferon-g; iNOS,inducible NO-synthase; NO, nitric oxides; PTK, phototherapeutic keratectomy; RNI, reactive nitrogen intermediates.

Part of the results described in this letter were presented as a poster (P-MO-142) in the Section Refractive Surgery and Refraction (P-30) at

the World Ophthalmology Congress 2010 (WOC®2010, XXXII International Congress of Ophthalmology), Berlin, Germany, 5–9 June 2010.

Letters to the Editor 277

© 2011 The AuthorsClinical and Experimental Ophthalmology © 2011 Royal Australian and New Zealand College of Ophthalmologists

as described elsewhere.3 The statistical analysis wasperformed by using the unpaired Student’s t-test and thePearson’s correlation coefficient (PCC).

One hour after PTK treatment, the expression of IFN-gand iNOS as well as that of NO/RNI were all higher thanin the untreated controls, but lower than 12 h after treat-ment (Table 1). The increases observed between 1 and12 h after PTK were all statistically significant (P = 0.0491for IFN-g, 0.0105 for iNOS and 0.0096 for NO/RNI,Table 1). The gene expression of iNOS could be detectednot only in epithelial, endothelial and infiltrating cells,but also in the keratocytes of the corneal stroma (Fig. 1).Interestingly, the gene expression of iNOS was found toshow a direct correlation with the concentrations of IFN-g(PCC = 0.907) and NO/RNI (PCC = 0.984) (Table 1). Adirect correlation between the concentration of IFN-g andthe activity of iNOS has previously been described in dif-ferent biological systems,3 including the aqueous duringcorneal graft rejection.4 Whereas 24 h after PTK treatment,

the expression of iNOS and the accumulation of NO/RNIremained higher than in the controls, slight decreasescould be observed, when compared with the correspond-ing values 12 h after PTK (Table 1). In a previous studyperformed with New Zealand white rabbits, in spite ofthe especially high NO levels observed for naïve rabbits,the NO levels detected in the aqueous of treated animalsshowed increases of 33–68% between 4 and 24 h afterexcimer laser ablation,5 which are comparable with thedetected NO/RNI increases of 69% between 1 and 12 hafter PTK in the plasma of Wistar rats (Table 1).

Taken together, after PTK treatment, the expression ofIFN-g and NO/RNI in plasma as well as the mRNA geneexpression of iNOS in corneal cells were all significantlyincreased, especially 12 h after treatment, which may playa relevant role in wound healing after laser treatment.

ACKNOWLEDGEMENTS

The authors are very grateful to Andrea Zelmer, Charité-UM, BMFZ-CVK, Berlin, Germany, for an excellent tech-nical assistance. JP was partially supported by the RudolfSiedersleben-Otto Wolff Foundation, Cologne, Germany,and TN-T was fully supported by Deutscher AkademischerAustausch Dienst (DAAD), Berlin, Germany. In all thedescribed animal experiments, the ‘Principles of laboratoryanimal care’ (NIH), the OPRR Public Health Service Policyon the Humane Care and Use of Laboratory Animals andthe US Animal Welfare Act, as amended, were followed, aswell as specific national laws where applicable.

Javier Prada PhD1,2 and Thanh Ngo-Tu MD PhD2,3

1Biomedical Research Center, Charité-UniversitätsmedizinBerlin, Campus Virchow Klinikum, Humboldt

University of Berlin, 2Department of Ophthalmology,Charité-Universitätsmedizin Berlin, Berlin, Germany; and

3Department of Microbiology, University of Hanoi,Hanoi, Vietnam

Received 7 September 2010; accepted 8 September 2010.

REFERENCES

1. O’Brien TP, Li Q, Ashraf MF, Matteson DM, Stark WJ,Chan CC. Inflammatory response in the early stages ofwound healing after excimer laser keratectomy. ArchOphthalmol 1998; 116: 1470–4.

2. Pettit GH, Ediger MN, Hahn DW, Landry RJ,Weiblinger RP, Morehouse KM. Electron paramagneticresonance spectroscopy of free radicals in corneal tissuefollowing excimer laser irradiation. Lasers Surg Med1996; 18: 367–72.

3. Prada J, Mueller S, Bienzle U, Kremsner PG. Up-regulation of reactive oxygen and nitrogen intermedi-ates in Plasmodium berghei infected mice after rescuetherapy with chloroquine or artemether. J AntimicrobChemother 1996; 38: 95–102.

4. Nicholls SM, Dick AD. Lack of interferon-gamma syn-thesis in aqueous humor during corneal graft rejectioncorrelates with suppressed nitric oxide production bymacrophages. Invest Ophthalmol Vis Sci 2008; 49: 4923–30.

Figure 1. Gene expression of iNOS in corneal cells of Wistarrats after PTK, as detected by mRNA in situ hybridizations usingan iNOS-specific gene probe. (a) iNOS hybridization patternobserved in corneal cells 12 h after PTK treatment, showing avery extended positive reaction (+++, hybridization score: 4.0).(b) iNOS hybridization pattern observed in corneal cells 24 h afterPTK treatment, showing an extended positive reaction (++,hybridization score: 3.0). en, corneal endothelial cells; iNOS,inducible NO-synthase; NO, nitric oxides; PTK, phototherapeutickeratectomy; st: corneal stroma cells.

278 Letters to the Editor

© 2011 The AuthorsClinical and Experimental Ophthalmology © 2011 Royal Australian and New Zealand College of Ophthalmologists

5. Adiguzel U, Bilhigan K, Ozdek SC, Sancak B, Hasanre-isoglu B. Nitric oxide levels of aqueous humor afterphotorefractive keratectomy. Eur J Ophthalmol 2004; 14:100–5.

Glaucoma progression associatedwith altered cerebral spinal fluidlevels of amyloid beta andtau proteins

Several studies have revealed links between Alzheimer’sdisease (AD) and glaucoma.1 This report describes a glau-coma patient with medically controlled intraocular pres-sure (IOP) who experienced disease progressionconcomitantly with the onset of mild cognitive impairment(MCI) and positivity for cerebral spinal fluid (CSF)markers of AD.

In 2005 a 69-year-old man presented with reducedvisual acuity in the left eye. He had no history of hyper-tension, diabetes, cerebrovascular problems or cognitiveimpairment, but the family history was positive forglaucoma. Best-corrected visual acuity in the right eyewas 0.1 logMAR; the left eye could barely perceive light.IOPs were 25 mmHg (right eye) and 28 mmHg (left eye).Exam of the anterior chamber with the gonioscopic lensrevealed an open irodocorneal angle. Visual field testing(Humphrey sita standard 30–2) of the right eye revealedstage 4 glaucoma (Brusini Glaucoma Staging System 2,GSS2) (Fig. 1). The optic nerve head of the right eyepresented advanced cupping with total loss of superior,inferior and temporal disc tissue and with nasal displace-ment of the blood vessels (Fig. 2a). In the left eye,all the neural rim was destroyed, and the opticnerve head appeared white and deeply excavated(Fig. 2b).

Advanced open-angle glaucoma was diagnosed andtreatment started with timolol 0.5% BID and travoprost(both eyes). Follow-up visits every 6 months for 4 yearsrevealed IOPs < 16 mmHg and no change in the visualfields. Between February and October 2009, he devel-oped progressive visual loss in the right eye (0.7logMAR), and worsening of the visual field to GSS2stage 5 (Fig. 1) although the IOP was 14 mmHg. Becausehis wife reported concomitant onset of memory deficits,the Mental Deterioration Battery was performed and MCIwas diagnosed. Routine blood work including B12 andfolate levels, screening for vasculitis (erythrocyte sedi-mentation rate (ESR), C-reactive protein, antinuclearantibodies, rheumatoid factor) and syphilis serology werenormal. Blood pressure monitoring for episodes of sys-temic hypotension, electrocardiogram and epiaortic ultra-sonography were also unremarcable. Magnetic resonanceimaging with gadolinium found normal-appearing visualpathways. CSF markers of neurodegenerative dementiawere assessed with commercial ELISAs (Innogenetics,

Ghent, Belgium). The results were consistent with AD:decreased amyloid beta (Ab)1–42 (226 pg/mL) and elevatedlevels of total and phosphorylated tau (t-tau and p-tau)(655 and 105 pg/mL, respectively).2

An increased incidence of primary open-angle glau-coma has been reported among AD patients,1 and a Japa-nese population study3 found that almost all such casesare the normal-tension form, which suggests the involve-ment of causative factors unrelated to IOP. Glaucoma alsoseems to progress more rapidly in AD patients than inother groups.4 In addition, in rats with experimentalocular hypertension, activation of caspase 3 in the retinalganglion cells has been shown to cause cleavages of theamyloid precursor protein that generate Ab and otherneurotoxic species.1 This is consistent with reports thatblocking the effects of Ab prevents retinal ganglion cellapoptosis.5 Recent evidence indicates that altered CSF cir-culatory dynamics can reduce the clearance of both Aband tau,2 and decreased Ab and increased t- and p-taulevels in the CSF have been shown to predict progressionfrom cognitive normality to MCI or AD-type dementiaand from MCI to AD. Cut-off values were 550 (95% CI531–570) ng/L for Ab, 375 (325–405) ng/L for t-tau and52 (48–56) ng/L for p-tau.2 Our experience with thepatient described here suggests that in advanced glau-coma, the onset of cognitive impairment may be associ-ated with progression to end-stage glaucoma, regardlessof IOP levels. Apart from predicting imminent dementia,the altered CSF levels of Ab and tau proteins observed inthis case might also reflect increased risk for progressionof glaucoma to the stage of visual loss. It is also possiblethat altered CSF circulatory dynamics in this case reducedneurotoxin clearance along optic nerves in the subarach-noid space and that deposits/aggregates of tau and/orother toxic molecules may have contributed to the glau-coma progression (see Killer et al. 20086). This seems tobe the first report of severe glaucoma progression associ-ated with CSF alterations indicative of AD, and itrequires confirmation in large studies.

Carlo Nucci MD PhD,1 Alessio Martucci MD,1

Alessandro Martorana MD,2 Giulia MariaSancesario MSc3 and Luciano Cerulli MD1

Departments of 1Ophthalmology, 2Neuroscience, and3Clinical Biochemistry and Molecular Biology,

University of Rome Tor Vergata, Rome, ItalyReceived 10 September 2010; accepted 14 September 2010.

REFERENCES

1. Wostyn P, Audenaert K, De Deyn PP. Alzheimer’sdisease and glaucoma: is there a casual relationship? BrJ Ophthalmol 2009; 93: 1557–9.

2. Perrin RJ, Fagan AM, Holtzman DM. Multimodal tech-niques for diagnosis and prognosis of Alzheimer’sdisease. Nature 2009; 416: 916–22.

3. Tamura H, Kawakami H, Kanamoto T et al. High fre-quency of open-angle glaucoma in Japanese patientswith Alzheimer’s disease. Neurol Sci 2006; 246: 79–83.

Letters to the Editor 279

© 2011 The AuthorsClinical and Experimental Ophthalmology © 2011 Royal Australian and New Zealand College of Ophthalmologists