the cellular resistance to pH changes in iso-osmolar tissue culture media. We worked out a systematic series of measurements of the pH neutralizing action of any type of rinsing fluid available in the market. These series included tap water, saline, borate, phosphate in different formulations, and Diphoterine®.

6.2  Mechanisms of Diffusion

in Rinsing Therapy

If any corrosive touches the corneal surface, it first breaks into the tear fluid film; later, the tight junctions of the epithelium have to be broken after the epithelium is denuded from the surface. The propagation through the corneal stroma is limited by the architecture of homogenous lamellae. To classify the speed and variation of propagation into the cornea, special measurements are required. These measurements rely on the first change of pH of the aqueous humor which is close to the endothelium. They are made with a pH electrode. By these measurements, in eye burns with 50 mL of 2M NaOH, we have found a relatively stable speed of diffusion through the corneal stroma of 5–8 mm/s. By this, the time of damage of the endothelium starting with a severe rising of pH can be confirmed to be around

80–100 s from the initial eye burn of the corneal surface. The propagation of the corrosive with intraocular pH change follows a stable pattern with a consecutive severe pH change throughout all layers of the cornea. The work of Pospisil et al. [10] opens a theoretical model that explains the introduction of corrosives into the eye and the outflow of these materials by the diffusion. The model still lacks the influence of distribution coefficient changes due to the alteration by severe corrosives and the influence of other substances introducing chemical interaction like neutralization, amphoteric binding, or precipitation of unsoluble salts. We can observe by highresolution real time optical coherence tomography (OCT) the diffusion of hydrofluoric acid into the cornea (Fig. 6.1). The mechanistic understanding of the inflow is relatively completed, but the decontamination is still not understood by means of mathematical models. This necessitates more work in the near future.

6.3  Osmolar Effects in Rinsing Therapy

The effects of osmolarity on cells are well known from physiology. Semipermeable membranes allow a water flux depending on the number of solved ions. Therefore, a cell with its own tissue osmolarity of about

Fig. 6.2  Healthy corneal stroma, aqueous fluid, and corneal stroma osmolarities after prolonged 16 day treatment with phosphate buffer (Isogutt®), saline solution, and Diphoterine®

420 mOsmol/kg in the cornea [11] will swell if tap water is applied to a denuded stroma, where epithelial tight junctions normally prevent water influx [12]. This is the deeper origin of well-known surgical corneal swelling due to hypoosmolar rinsing fluids in damaged epithelial cells. Here lies the origin of Descemets folds and corneal clouding directly after eye burns and water flushing [13]. The osmolarity of highly dissociated corrosives is directly dependent on their molarity and dissociation constants. Exceeding concentrations of dissociating fluids of more than 420 mOsmol/kg will result in a water loss directly due to the trauma and a consecutive severe edema after flushing with hypoosmolar water [8]. For cellular populations loaded with ions over their normal levels, there is a considerable evidence that low osmolarity will destroy cell borders.

There are several most interesting observations concerning osmolarity. We have found that there is an important difference of osmolarity between the hyperosmolar stroma with 420 mOsmol/kg and the extra and intraocular fluids with lower osmolarities of about 320 mOsmol/kg. This results in an inversion of the water flux, when barriers like endothelium and epithelium are damaged. The immediate result of any membrane damage is that water starts to flow from the outside into the corneal stroma. This results in a corneal edema with turbidity. This is an indirect proof of the pumping function of the endothelium.

In several experiments, we have measured the corneal stroma and intracameral osmolarity by means of cryo milling and redilution of the stroma of the burnt cornea and by direct measurements of the intraocular fluids.

We have observed the following pattern (Fig. 6.2) of decrease of the tissue osmolarity after eye burns within the corneal stroma. This confirms the clinical finding of severe edema of the stroma and was sustained by work of Kompa et al. [8], who have found the alteration of the corneal swelling and corneal osmolarity by the eye burn itself and changes due to different fluids used for rinsing of the cornea thereafter.

All burnt corneal stroma show lower osmolarity due to the loss of ions by continuous rinsing. The higher osmolar Diphoterine® adds some ions to the burnt corneal stroma, but its osmolarity is still much lower than the one of the aqueous humor. It is obvious that there is no change in osmolarity in aqeous humor in healthy and in burnt eyes due to autoregulation of this milieu by the ciliary body secretion. The considerable differences in tissue osmolarity indicate that there will be a strong fluid uptake into the corneal tissue, resluting in opacity. This indicates a severe water uptake and a lack of the endothelial pumping function.

This severe water uptake is confirmed by the next figure (Fig. 6.3) showing the hydration of the cornea after burn that is kept untreated for up to 7 days or respectively treated by three times daily rinsing of the eye with phosphate buffer (Isogutt®), saline solution, or Diphoterine® solution for 16 days.

The untreated eye burns result in a severe and high water uptake. With rinsing therapy, this does not really change to a large extent after 16 days. The lower hydration in the phosphate group is due to the calcification and loss of stromal ground substance that is able to swell.

Fig. 6.3  Water contents of healthy and burnt cornea. The hydration level is obtained as follows: (dry mass (g) + water (g))/ water (g)

Treatment type

Corneal edema in %

Fig. 6.4  Osmolarity of different rinsing solutions

40

Tap water 3 mOmol/kg

35

NaCl 1,200 mOsmo/kg 30 NaCl 800 mOsmol/kg

NaCl 400 mOsmol/kg

25

20

15

10

5

0

Rinsing fluid

The reversal of the osmolarity-caused corneal swelling after eye burns is not possible by increasing osmolarities in the rinsing fluids as tried with saline solutions with different osmolarities (Fig. 6.4). The result of this experiment is that, even with the highest osmolarities, the reversal of the stromal edema is not

completely possible, but a higher amount of initial swelling can be prevented. Even with 1,200 mOsmol/ kg rinsing, the thickness of the burnt cornea increases by 5%, thus showing the uptake of water.

Osmolar effects lead to cell lysis as we can demonstrate in epithelial (ARPE19) and mesenchymal

cell (murine fibroblasts L929) cultures, thereby, in rinsing of cellular tissues that are immersed with 800 mOsmol/kg, which is twice as much the normal 360–420 mOsmol/kg in cell culture medium.

It has been found that the osmolar stress releases Interleukins from cells and has an intrinsic proinflammatory action.

The cell lysis that we observed in rinsing experiments liberates fragments of cells and necrotic material leading to fast inflammation.

In Fig. 6.5, tissue culture preincubated with 800 mOsmol (NaCl), shows cells of normal shape and function.

The same tissue culture after exposure to tap water, with blown-up cells after 60 s of water exposure is shown in Fig. 6.6.

After 120 s of exposure, it is shown with total cytolysis and necrosis (Fig. 6.7).

If any rinsing solution that is isotonic to blood such as 0.9% NaCl or RINGER lactate is applied, the result of cellular damage will be different. The cells blow up but keep their integrity.

Tissue culture incubated with RINGER lactate of 290 mOsmol is shown in Fig.6.8. Cells are blown up, but still, membranes are of good integrity (Fig. 6.8).

Higher osmolarities in early rinsing have effect on the integrity of cells and stabilize the cellular bodies against the water influx. This concept is realized by Diphoterine® of 820 mOsmol.

Tissue culture is preincubated with 800 mOsmol (NaCl) and contains cells of normal shape and function.