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International audience; Purpose: The authors present ultrastructural and histologic studies of femtosecond laser incisions in human cornea. Methods: We use a CPA femtosecond laser designed to match current instrumentation for femtosecond surgery, i.e. a Nd:Glass femtosecond laser at a 1065 nm wavelength. The repetition rate is adjustable from 1 to 10 KHz, with maximum pulse energy of 60 µJ. The human cornea samples are fixed to an anterior chamber system that can be moved in 3D by micrometric precision motors in front of a fixed objective delivering the fs laser pulses. Human corneas are provided by the French Eye Bank. Laser energy range for cornea treatment goes from laser induced optical breakdown threshold up to ten time this threshold. Ultrastructural and histologic are then obtained using transmission electronic microscopy TEM and scanning electron microscopy (SEM). Results: Observations of the cornea structures (i.e. epithelium, Bowman membrane, stroma, Descemet membrane and endothelium) presents no noticeable side damages like burned collagen or cells damage. Results does not vary with pulse energy (in the experimental energy range : 2 J/cm2 to 50 J/cm2) except for excessive delamination due to the shock wave propagation at high energy. Conclusions: The use of femtosecond laser pulses for keratoplasty implies use of energy higher than the one used for refractive surgery in transparent cornea. Our results demonstrate that cornea ultrastructure is not altered by this higher energy pulses opening the way to surgery in oedematous cornea.

International audience; Purpose: The interacting phenomena that happen between femtosecond lasers in edematous or pathological corneas are still not clearly known. Their characterization is fundamental to develop femtosecond laser use for penetrating and lamellar keratoplasty. The plasma threshold corresponds to the minimal energy that must be focalised into the cornea in order to induce ionization of matter. The possibility of making one corneal cut depends on plasma creation. We determined incident energy thresholds to obtain plasma in function of different corneal depths and hydrations. Methods: We use a Nd:Glass femtosecond laser, with a CPA system, and 1065 nm wavelength. The repetition rate is adjustable from 1 to 10 KHz, with maximum pulse energy of 60 µJ. The pulse delivery is done through an objective adapted on an optic table. The samples are fixed to an anterior chamber system and moved 3–dimensionally by micrometric precision motors. Human corneas are obtained from the French Eye Bank. Three corneal groups with 10 corneas each are organized depending on corneal pachymetry: Group 1: 100 µm +/– 75 µm, Group 2: 700 µm +/– 45 µm, Group 3: 500 µm +/– 25 µm. The control group is composed of silicon dioxide samples. The samples are analyzed in confocal microscopy of the experimental set up. Results: The incident energy threshold is similar in the control group and in the group 3; it ranges from 5 J/cm² on surface to 10 J/cm² at 500 µm depth. In cornea Groups 2 and 1, the incident energy threshold increases, respectively, of 5 J/cm² and of 10 J/cm² for each 100 µm of corneal depth that the laser beam travels through. Conclusions:In each given group, the incident energy threshold increases proportionally to the corneal thickness the laser beam traveled through. To the same corneal thickness, the threshold is higher in the most edematous corneas. The knowledge of these results is fundamental in order to determinate cut parameters and side effects in corneas with hydration above the normal.

International audience; Purpose: The authors present a microsurgery experimental system using Nd: Glass femtosecond laser. It is coupled to a confocal microscopy observation system to investigate intrastromal effects of the femtosecond laser. Methods: The femtosecond laser is a CPA system with a regenerative amplifier delivering pulses at a wavelength of 1.065 µm, with a pulse duration of 500 fs, an energy of 60 µJ for a repetition rate of 10 KHz. The amplified pulses power fluctuations are lower than 1%. Pulses repetition rate is adjustable up to 10kHz and can even be reduced to single shot. The laser beam has a Gaussian distribution shape TEM00. The obtainable peak power density is on the order of 10^15W/cm^2. The laser beam travels to the samples guided by one six–lens set and one magnifier. The last lens can be changed to adjust the focal distance from 2 to 5 cm and the focal spot from 1 to 4 µm. The samples are fixed to an anterior chamber system, which is driven by a micrometric precision motor system. The 3 dimensional movements are computer controlled. The laser energy is continuously monitor for measurements consistency. The experimental set up is also composed of a confocal microscope, which permits corneal sample analysis without removing them from the artificial chamber system. Results: The experimental set up permits complex geometry to be done. Penetrating and lamellar cuts could be performed in silicon dioxide and human corneal samples. The confocal microscopy observation system permits precise parameter control of the cutting system (cut shape, shot scanning and treatment depth). Conclusions: Applications for femtosecond laser are the refractive surgery and corneal keratoplasty. The development of concepts as sutureless keratoplasty needs an experimental system with large flexibility in order to work out useful cut parameters.

International audience; Purpose: When a femtosecond laser beam causes photodisruption, a cavitation bubble is produced. Many laser shots may be placed close together, and the induced cavitation bubbles coalesce to create a cut. Reduction of the bubble diameter is fundamental to improving the cut quality. Methods: A home–built Nd:Glass femtosecond laser is used with a CPA system and 1065 µm wavelength. The pulse repetition rate ranges from 1 to 10 KHz, with a maximum pulse energy of about 60 µJ. The laser delivery system works through an objective lens on an optical table. The samples are fixed to a 3–dimensional moving anterior chamber system, which is connected to a micrometric stepper motor. Human corneas are obtained from the French Eye Bank and are divided into 2 groups, according to their pachymetry: Group 1 (n=10): 1000 µm +/– 75 µm, and Group 2 (n=10): 700 µm +/– 45 µm. The control group is composed of silicon dioxide samples. The treated corneas are analyzed through confocal microscopy. Results: At plasma threshold, the average cavitation bubble diameter is: 6 µm +/– 7µm in the Group 1 corneas, 25 µm +/– 1.5 µm in the Group 2 corneas and 2 +/– 1 µm in the silicon dioxide samples. In group 2 corneas and silicon dioxide samples, the majority of laser pulses generated cavitation bubbles. In group 1 corneas, far fewer of the laser pulses induced cavitation bubbles. The time it takes for one cavitation bubble to disappear is: 90 +/– 10 seconds for a 25 µm cavitation bubble, 25 +/– 5 seconds for a 6 µm cavitation bubble, and 10 minutes in the silicon samples. Conclusions: In more edematous corneas, the cavitation bubble diameter is heterogeneous. This heterogeneity is due to laser beam attenuation by the corneal edema. This information suggests that, in the absence of laser parameter adaptation, the cut quality in edematous and pathological corneas will be less than in clear corneas.

PURPOSE: To investigate the involvement of the cornea during endotoxin-induced uveitis (EIU) in the rat and the effect of Ngamma-nitro-L-arginine methyl ester (L-NAME) as nitric oxide synthase (NOS) inhibitor, administered by iontophoresis. METHODS: EIU was induced in Lewis rats that were killed at 8 and 16 hours after lipopolysaccharide (LPS) injection. The severity of uveitis was evaluated clinically at 16 hours, and nitrite levels were evaluated in the aqueous humor at 8 hours. Corneal thickness was measured, 16 hours after LPS injection, on histologic sections using an image analyzer. Transmission electron microscopy (TEM) was used for fine analysis of the cornea. Transcorneoscleral iontophoresis of L-NAME (100 mM) was performed either at LPS injection or at 1 and 2 hours after LPS injection. RESULTS: At 16 hours after LPS injection, mean corneal thickness was 153.7+/-5.58 microm in the group of rats injected with LPS (n=8) compared with 126.89+/-11.11 microm in the saline-injected rats (n=8) (P < 0.01). TEM showed stromal edema and signs of damage in the endothelial and epithelial layers. In the group of rats treated by three successive iontophoreses of L-NAME (n=8), corneal thickness was 125.24+/-10.36 microm compared with 146.76+/-7.52 microm in the group of rats treated with iontophoresis of saline (n=8), (P=0.015). TEM observation showed a reduction of stromal edema and a normal endothelium. Nitrite levels in the aqueous humor were significantly reduced at 8 hours by L-NAME treatment (P=0.03). No effect on corneal edema was observed after a single iontophoresis of L-NAME at LPS injection (P=0.19). Iontophoresis of saline by itself induced no change in corneal thickness nor in TEM structure analysis compared with normal rats. CONCLUSIONS: Corneal edema is observed during EIU. This edema is significantly reduced by three successive iontophoreses of L-NAME, which partially inhibited the inflammation. A role of nitric oxide in the corneal endothelium functions may explain the antiedematous effect of L-NAME.

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