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Файл:Ординатура / Хирургия / Библиотека им академика М.И. Перельмана / Книга_5915_Библиотеки_им_академика_М_И_Перельмана
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choroid
retina
vitreous
depth
resolution
V.F. reading
II
III
I
Fig. 3.3 A schematic drawing of the focal diamond advancing through the choroidal–retinal structure
with the resulting fluorescence records intensity as a symmetrical curve (Zeimer et al. 1982)
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Fig. 3.2 Development of spread function, Sx, by movement of the focal diamond along the optical
axis, x, through the cornea, Q, with a thickness of dq (Joshi et al. 1996)
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Fig. 3.4 The fluorophotometer excitation and emission beam are focused through the ocular
structures. As the beam is advanced through the eye, the fluorescence values are used to plot the
ocular fluorescence profile of the eye
3.2 Normal Human Subject and Rabbit Ocular Fluorescence
The autofluorescence of the crystalline lens increases with age (Chang and Hu 1993;
Chang et al. 1995). Figure 3.5 is an autofluorescence scan of a 57-year-old male
with the crystalline lens fluorescence of 300 ng/mL and the tear/cornea peak at
10 ng/mL. Figure 3.5a was captured with the anterior chamber lens which provides
a greater resolution to separate the tear/cornea peak from the lens peaks. Figure 3.5b
was captured with the standard objective lens. In contrast, the fluorescent profile for
the young rabbit (<6 months old) illustrated a different tear/cornea to lens ratio
(Fig. 3.6). The rabbit crystalline lens fluorescence is 2 ng/mL and the tear/cornea
peak at 3.5 ng/mL.
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3 Fluorophotometry for Pharmacokinetic Assessment
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Fig. 3.5 The ocular scan with the anterior chamber lens plots the natural fluorescence through the
anterior segment of the eye of a 57-year-old male. (a) Captured with the anterior chamber objective
lens. (b) Captured with the standard objective lens
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Fig. 3.6 The ocular scan plots the natural fluorescence through the full length of a young
(<6 months) rabbit. (a) Captured with the anterior chamber objective lens. (b) Captured with the
standard objective lens
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3 Fluorophotometry for Pharmacokinetic Assessment
1
%
Tear turnover 100(1 ).
min
a
e== −
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3.3 Fluorophotometry Applications
The fluorophotometer has been used to determine tear flow rates (Occhipiniti et al.
1988), normal corneal epithelial permeability in the human (de Kruijf et al. 1987;
Schalnus and Ohrloff 1990) and rabbit (McCarey and al Reaves 1995), corneal endothe-
lial permeability (Mishima and Maurice 1971; Ota et al. 1974), aqueous humor turnover rate (Jones and Maurice 1966), blood aqueous barrier permeability (van Best et al.
1987), crystalline lens transmission (van Best et al. 1985), and blood–retinal barrier
(Cunha-Vaz et al. 1975; Grimes et al. 1982). The fluorophotometer provides a tool to
access ocular toxicity as an alteration in normal physiological parameters. The effects
have been measured for drugs altering basal tear turnover (Kuppens et al. 1992, 1994),
contact lenses on corneal epithelial permeability (Boets et al. 1988), contact lenses on
endothelial permeability (Ramselaar et al. 1988; Gobbels and Spitznas 1992; and drug
effect on the permeability of the blood–retinal barrier (Cunha-Vaz et al. 1975; Tsuboti
and Pedersen 1987). Noninvasive ocular fluorophotometry can provide a sensitive
indicator of the ocular toxicity effects of a drug, preservative, surgical procedure, etc.
on the normal physiology parameters of epithelial permeability, McCarey and Reaves
1997), endothelial permeability, aqueous turnover rate, etc.
3.3.1 Tear Turnover Rate (%/min)
The fluorophotometer can be used to measure the basal or reflex tear turnover
rate noninvasively. Kuppens et al. (1992) used 1 mL of a 2% solution of sodium
fluorescein and scanned the eye for 30 min at 1.5 min intervals to measure the
basal tear turnover rate. An accurate measure can only be performed by carefully
avoiding the reflex tear response. In my laboratory, I used either 1 mL of a 0.2%
sodium fluorescein or 10 mL of a 0.2% sodium fluorescein. The additional volume to the tear will not overwhelm the natural tear and cause the extra volume to
produce erroneous data for the first 7 min. The tear/cornea peak fluorescence at
2-min intervals between 5 and 30 min after the tear application is plotted on a
linear scale relative to post-fluorescein application (Fig. 3.7). An exponential
curve is fit to the data to calculate the curve coefficients (intercept a0, slope a1,
correlation coefficient r).
After 30 min, the fluorescein concentration is so low that six to eight drops of BSS
will clear the tear film of applied fluorescein. The added fluorescein will be cleared
after an additional 20 min; at which time the eye can be evaluated for tear turnover
again.
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Fig. 3.7 Following a single topical application to the conjunctiva of 1 mL of 0.2% sodium
fluorescein, the human subject had tear/cornea fluorophotometer scans between 6 and 29 min postapplication. The exponential decrease in fluorescein resulted in a 9.3%/min tear turnover
3.3.2 Corneal Epithelial Cell Layer Permeability Methodologies
There are three basic techniques to quantify the fluorescein uptake in the cornea for
epithelial cell layer permeability determination.
1. Eye bath technique to measure epithelial permeability: A 20 mL eye bath of 1%
sodium fluorescein is maintained against the eye for 3 min. After the fluorescein
exposure, the eye receives five 30 s rinses with a balanced salt solution. During the
next hour four consecutive fluorophotometric scans are performed every 10 min
(de Kruijf et al. 1987; Boets et al. 1988; Ramselaar et al. 1988). The epithelial cell
layer permeability coefficient can be calculated.
2. Single drop technique to measure fluorescein uptake in the cornea: A 20 mL drop of
2% sodium fluorescein is instilled onto the cul-de-sac. Forty-five minutes later the
fluorescein concentration is measured in the cornea. This measurement is referred
to as the F45 (Berkowitz et al. 1981; Gobbels et al. 1989; Gobbles and Spitznas 1989;
Chang and Hu 1993). The F45 provides relative epithelial cell layer permeability.
3. Single drop technique to measure epithelial permeability: A 1.0 mL drop of 10%
sodium fluorescein is instilled onto the cul-de-sac. A tear sample is measured to
determine the tear-diluted fluorescein concentration. Four minutes later fluorophotometric scans are performed every 90 s for 15 min, then at 45 min after fluorescein
application (Gobbels and Spitznas 1992). A modification of this technique was
published by Joshi et al. (1996). They used a 2.0 mL drop of 0.75% sodium fluores-
cein instilled onto the cul-de-sac and requested the patient to blink vigorously. The
fluorescence of the tear film was followed for 20 min until it became comparable
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3 Fluorophotometry for Pharmacokinetic Assessment
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TO C
B
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CF F
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Ft
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to that in the cornea. Scans were repeated as rapidly as possible for 8 min, then
every 2 min for 20 min. The fluorescein was flushed from the cul-de-sac for 1 min.
Two more scans were performed to determine corneal stromal fluorescein concentration. The epithelial cell layer permeability coefficient can be calculated.
3.3.3 Eye Bath Technique
de Kruijf et al. (1987) were the first to report the use of an eyebath to deliver a known
concentration of fluorescein to the cornea of human subjects. The epithelial permeability is estimated by dividing the resultant increase in corneal fluorescein by the
product of the bath concentration and bathing time. Although the bath technique
provides an easy method to estimate human in vivo epithelial permeability, its clinical
use is limited because the method of fluorescein application is difficult for many
subjects to tolerate and may lead to fluorescein staining of the skin. Despite the subject acceptance limitation, the technique is still being used (Tognetto et al. 2001).
Noninvasive fluorophotometry bath technique is easily performed with the
unanesthetized rabbits with n = 6 per experimental group. The stock sodium fluorescein
solution (376.3 mol. wt.) is 0.075% (750,000 ng/mL) dissolved in BSS. Store the fluorescein solution at 4°C and protected from light. Check daily the concentration of the
corneal sodium fluorescein bathing solution by diluting the stock solution 1:1,600 to
yield 469 ng/mL. Corneal autofluorescence should be determined from the average of
three fluorescence values on either side of the peak value from the anterior segment scan
of the eye. The values will be 3–5 ng/mL for the rabbit. Bathe the cornea in 0.075%
sodium fluorescein in vivo by the following technique. Place the unanesthetized rabbit
on paper towels. Position yourself behind the rabbit while holding its lower eyelid open
in a cup-like position. Fill the cul-de-sac with 0.075% sodium fluorescein at 33°C for
3 min. Add fresh sodium fluorescein as needed to keep the cornea covered. Make sure
the eyelids are pulled away from the cornea to permit good bathing of the corneal epithelium. After the 3-min sodium fluorescein exposure, wick off the sodium fluorescein
with a tissue. Flush away remaining fluorescein with 40 mL of 33°C BSS applied with
a disposable pipette and bulb. Examine the eye with a slit lamp and blue filtered light.
The normal cornea should not demonstrate general stromal uptake of sodium fluorescein. Note any staining pattern and location on the data form. Measure the corneal
sodium fluorescein concentration with the Fluorotron Master Anterior Segment Adaptor.
Measure and save three to six repeated readings immediately after BSS rinse. Measure
the corneal thickness with an ultrasonic pachymeter. Print the data files from the
Fluorotron. Select the peak corneal sodium fluorescein concentration for each repeated
scan. Epithelial permeability analysis uses the following formula:
= corneal thickness
C
T
FO = stromal fluorescence
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Fig. 3.8 The in vivo noninvasive corneal epithelial cell layer permeability to carboxyfluorescein
was determined in the rabbit eye (McCarey and al Reaves 1995)
FC = autofluorescence
FB = fluorescein bath
t = bath duration
FO and FC are adjusted for spatial resolution of the focal diamond scanning the thin
cornea
The normal rabbit corneal epithelial cell layer permeability to carboxyfluorescein was measured by McCarey and al Reaves (
1995). A skewed bell-shaped
histogram (Fig. 3.8) has epithelial cell layer permeability to carboxyfluorescein of
0.0646 ± 0.00700 nm/s.
3.3.4 Single Drop Technique to Measure Epithelial Permeability
There have been many strategies to assess corneal epithelial permeability by applying
a single drop of fluorescein to the ocular surface and estimating the amount of fluorescein remaining in the cornea at a specific time after application. Although simple to
perform, it is difficult to know the concentration of fluorescein bathing the epithelium.
The tear turnover continuously reduces the concentration of fluorescein available for
penetration. Variations in eyelid blink rate (tear mixing) and variations in tear production amplify the test accuracy. In dry eye patients with abnormal tear factors the accuracy of the F45 style test (relative fluorescein uptake at 45 min) is even further stressed.
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Yet, recent reports in the literature have used the technique to measure corneal
epithelial barrier function with aging (Chang and Hu 1993). Yokoi et al. (1998) applied
3 mL of 0.5% sodium fluorescein into the lower cul-de-sac with a 20 mL BSS Plus
rinse 30 min later (F30 relative fluorescein uptake at 30 min). The corneal fluorescein
uptake was determined from ten fluorophotometer scans. The control and treatment
groups were compared by the corneal fluorescein uptake in nanogram per milliliter.
The authors were able to use the technique to reveal subclinical ocular surface
epithelial cell layer problems in atopic dermatitis with blepharoconjunctivitis. The F30
do not provide an epithelial permeability coefficient.
and F
45
Gobbles and Spitznas (1989) attempted to improve the single drop technique by
accounting for individual tear turnover rates and changing concentration gradients.
They estimated permeability by dividing the increase in stromal fluorescein from
baseline autofluorescence levels 45 min after instillation by the tear film fluorescein
concentration integrated at the examination time of 45 min. The investigators failed to
consider the fluorescein trapped in the cul-de-sac which can be a depot for the dye.
Joshi et al. (1996) improved on Gobbels technique by adding a saline rinse
20 min after instillation of the initial fluorescein drop. Even with this modification,
the technique yielded human corneal epithelial values six times greater than the eye
bath technique. This would indicate that an unknown methodological problem may
exist. The basic single drop technique described by Joshi et al. cannot be used with
confidence until certain issues are resolved. It is important to highlight the observations from the Joshi et al. (1996) publication. High concentrations of fluorescein
were required to achieve a sufficient penetration into the cornea, and this led to an
error in estimating the tear film concentration of fluorescein which may be beyond
the linear range of the fluorophotometer. The Fluorotron Master (OcuMetrics,
Mountain View, CA) is only linear for 1–2,000 ng/mL before quenching reduces the
reliability. Figure 3.9 is reproduced from Joshi et al. (1996) to demonstrate the
effects of fluorescein quenching of a 2 mL drop of 0.5 and 1.5% fluorescein. The
explanation for the difference in the two curves in Fig. 3.8 is fluorescein quenching
at the higher fluorescein concentration.
In Fig. 3.10, the points on the straight line correspond to the fluorescein levels to
be expected in the tear if 2 mL drops of different concentrations were instilled and
the fluorescein was diluted three times in the tear film by already present tear. Thus,
a 1.5% drop should give rise to an initial fluorescein level corresponding to point A,
but because of absorption and more serious quenching; its value is depressed to A¢.
Similarly, if a drop of 0.5% fluorescein is applied, a fluorescein concentration at B
should be achieved, but the fluorophotometer will read the fluorescein concentration
at B¢. The dashed line is the relationship for the absorption of light by fluorescein
calculated in the algorithm used by the Joshi et
al. technique.
The data suggest that the maximum eye drop concentration that gives reliable
results is approximately 0.75% fluorescein. The scans should be taken at the same
time of day to avoid potential diurnal variations (Webber et al. 1987). Joshi et al.
(1996) preferred a 2 mL drop of 0.75% fluorescein. They found no correlation
between tear turnover and permeability (Table 3.1), which suggests that no systemic
error is introduced by the proposed algorithms.
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B.E. McCarey
DROP CONC (%)
Fluorescent Intensity (Volts)
Tear Concentration (% Fluorescein)
0.25
0.01 0.1 1
1
10
0.75
1.5
0.5
B
B'
A'
A
0
0510 15
.50%
1.50%
20 25
10
20
Fluorescence, a
d
(arbitrary units)
Time after instillation (min)
30
40
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Fig. 3.9 Contours of tear film fluorescein after instillation of a 2 mL drop of two concentrations of
fluorescein (Joshi et al. 1996)
Fig. 3.10 Relationship between tear film concentration and apparent fluorescein. The thin straight
line represents a theoretical linear relationship between fluorescein and concentration. The dashed line
is the relationship for the absorption of light by fluorescein calculated in the algorithm used by the
Joshi et al. technique
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