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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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3 Fluorophotometry for Pharmacokinetic Assessment
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Fig. 3.13 The fluorescein from either a suprachoroidal (top row) or intravitreal injection (bottom
row) must diffuse (left column at 0 h, middle column at 1 h, and right column at 4 h) to the central
vitreous before the fluorophotometer can measure the fluorescence)
to as tailing. After 4 h, fluorescein has diffused out of the retina and into the vitreous.
As the vitreous fluorescein reaches the optical axis of the eye, the fluorophotometer
scan measures an increase in the vitreous fluorescein (Fig. 3.13, top row, right column).
The cornea, aqueous humor, and lens fluorescence have not increased.
3.6 Retrobulbar Fluorescein Injection
A series of rabbits received retrobulbar injections to demonstrate the fluorescein
distribution in the eye. The retrobulbar injection of sodium fluorescein (100 mL of
25 mg/mL) was through the lower eyelid of NZW rabbits with 3–4 kg body weight.
Care was taken to avoid penetrating the globe and the conjunctiva. The Fluorotron
Master fluorophotometer (OcuMetrics, Mountain View, CA) with the standard
objective lens was used to scan the fluorescence through the entire globe to measure
tissue fluorescence (ng/mL).
Eight minutes after the retrobulbar injection of fluorescein, the cornea and retinal
florescence increased significantly. Relative to the autofluorescence in Fig. 3.14a,
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Fig. 3.14 The natural fluorescence of the ocular tissue of the rabbit is <6 ng/mL (a). The midvitreous value (4
peak. Eight minutes after the retrobulbar injection of fluorescein, the ocular fluorescence is greatly
increased (b). The corneal peak value is 230 ng/mL and the retinal peak is 254 ng/mL
ng/mL) was considered to be located 37% from the retina to the cornea/aqueous
the retinal fluorescein increased may be attributed to a vascular increase in fluorescein
(Fig. 3.14b). The corneal increase may be caused by fluorescein leaking from the
injection site or the conjunctival vessels into the tear film.
The fluorophotometer scans taken from the same unanesthetized rabbit 30 min postret-
robulbar injection shows a continual rise in retinal fluorescence with tailing into the
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Fig. 3.15 (a) Captured at 30 min postretrobulbar injection. (b) Captured at 90 min postretrobulbar
injection. The aqueous humor peak has been masked by the large retinal fluorescence
vitreous (Fig. 3.15a). The corneal did not have the same relative increase in fluorescence.
There is a slight increase in aqueous humor fluorescence, which is the secondary peak
adjacent to the corneal peak. By 90-min postretrobulbar injection, the tailing curve from
the large retinal fluorescence peak has masked the aqueous humor peak (Fig. 3.15b).
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Fig. 3.16 (a) Captured at 180 min postretrobulbar injection and (b) was captured at 420 min
postretrobulbar injection
The character of the retinal, vitreous, and aqueous fluorescence curves have not
changed, but only their magnitude at 180 min following the retrobulbar injection
(Fig. 3.16a). By 420 min postinjection (Fig. 3.16b), the magnitude of the ocular
fluorescence has decreased sufficiently to observe normal lens fluorescence, but the
vitreous retained the fluorescein. There is still an above-baseline fluorescence in the
aqueous humor.
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A sub-conjunctiva injection of sodium fluorescein resulted in a profile of sodium
fluorescein uptake and loss that was comparable to the retrobulbar-injected eyes.
The major difference was the sodium fluorescein leaked into the tear film through
the injection site in the conjunctiva. This caused direct and excessive corneal tissue
uptake of sodium fluorescein.
Experimental data in Figs. 3.14–3.16 were repeated in five rabbits to generate
summary plots in Fig. 3.17 with the anesthetized rabbit (in vivo). To assess the
Fig. 3.17 The anesthetized rabbits received retrobulbar injections of sodium fluorescein (a). The
contralateral eye did not receive retrobulbar injections (b). The baseline values for cornea, vitreous, and retina are indicated by dashed lines (n = 5, mean ± standard deviation)
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Fig. 3.18 The euthanized rabbit has received a retrobulbar injection of sodium fluorescein (a).
The contralateral eye did not receive a retrobulbar injection (b). The baseline values for cornea,
vitreous, and retina are indicated by dashed lines (n = 5, mean ± standard deviation)
effect of vascular circulation on the uptake and release of a retrobulbar fluorescein
injection, a series of euthanized rabbits (in situ) received retrobulbar injections
(Fig. 3.18). The retrobulbar fluorescein injection concentration was 100 mL of
25 mg/mL. The mid-vitreous values were defined as the fluorescence value is 37%
from the retina to the corneal aqueous peak:
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Cornea Peak value
Lens Peak value
Vitreous Peak value at mid-vitreous (37% from
the retina to the cornea peak)
Retina Peak value
In Fig. 3.17, the anesthetized rabbits were followed for 420 min following the
retrobulbar injections. The vitreous and retina fluorescein peaked early: 60
min
for vitreous and 30 min for retina. The corneal fluorescence increased and reached
a plateau at 120 min. Interestingly, the contralateral eye without the retrobulbar
injection of fluorescein mirrored the tissue fluorescence for the injected eye
(Fig. 3.17b). This observation supports the vascular connection between the two
eyes (Forster et al. 1979) The fluorescein exchange between the two eyes and the
loss of fluorescein from the ocular tissue reflects the presence of the blood flushing through the ocular tissue.
In the euthanized rabbit that has received a retrobulbar injection of sodium
fluorescein there is a slow uptake of fluorescein in equal concentration in the
cornea, vitreous, and retina (Fig. 3.18a). The contralateral eye mirrors the event
but at tenfold less concentration (Fig. 3.18b). The bodies were in muscular rigid-
ity by the 420-min examination. There was no indication of the ocular fluorescein concentrations returning to baseline. The results demonstrated uniform
cornea, mid-vitreous, and retina uptake for 3 h to a maximum of approximately
2,000 ng/mL. Interestingly, the contralateral control eyes had an uptake to
approximately 100 ng/mL.
3.7 Intravenous Fluorescein Injection In Vivo
The retinal/choroid and vitreous uptake and release of fluorescein can be used to
investigate in vivo intravenous drug uptake and release from the eye via various
modes of drug delivery. Intravenous injections of fluorescein were performed in a
third set of experiments. The ocular tissue autofluorescence prior to the injection is
plotted in Fig. 3.19.
The fluorescein intravenous injection (14 ng/kg) was in the marginal ear vein.
There was a rapid increase in the retinal fluorescein as the fluorescein was flush
through the retinal vasculature (Fig. 3.20). Fluorescein leaked from the retinal blood
vessels and penetrated into the vitreous but it is partially masked by the tailing concentrations of the retina. As the fluorophotometer sensor window moves through the
high retinal fluorescence, the resulting bell-shaped curve extends over the vitreous
section of the ocular fluorescence scan. The corneal fluorescein is most likely a
result of conjunctival fluorescein leakage into the tear film. The fluorophotometer
cannot distinguish between tear fluorescein and corneal fluorescein.
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Fig. 3.19 The ocular fluorescence through the full length of the eye is plotted prior to an IV injection of fluorescein. Note all fluorescence values are less than 10 ng/mL
A summary graph (Fig. 3.21) shows in vivo the cornea, vitreous, and retina and
release following the intravenous (IV) fluorescein injections in rabbits. The fluorescence values are in Table 3.2. There is a rapid flushing of the retina vasculature with
fluorescein; peaking 20 min after the injection and then rapidly dropping off. The
cornea also peaked in 20 min but did not rapidly release the fluorescein. The retinal
peak uptake was not achieved for 60 min. The characteristic uptake differences
between the retina, cornea, and vitreous can be explained by fluorescein being delivered and subsequently removed from the tissue by the vasculature. This resulted in
a maximum retinal sodium fluorescein value within 15 min. The cornea and midvitreous maximized at approximately 60 min. The sodium fluorescein peaks in the
cornea and mid-vitreous were two times greater in the IV-injected eyes than the
contralateral control eyes. The total ocular of sodium fluorescein uptake was
<0.0002% of the injected concentration. The retinal and mid-vitreous sodium fluorescein in both the retrobulbar-injected eyes and their control eyes returned to normal after 420 min, while the corneal sodium fluorescein remained elevated at
163 ng/mL in the retrobulbar-injected eyes and 53 ng/mL in the control eyes after
420 min.
The data support the difficulty of significant sodium fluorescein intraocular
uptake following periorbital injections. The ocular vasculature rapidly “washes”
away the sodium fluorescein and distributes it throughout the circulatory system,
including the noninjected contralateral control eyes.
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Fig. 3.20 The ocular fluorescence through the full length of the eye is plotted after an IV injection
of fluorescein in the anesthetized rabbit. (a) Captures at 1 min after the IV injection and (b) was at
9 min postinjection
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Fig. 3.21 Eye of animal with intravenous injection. The baseline values for cornea, vitreous, and
retina are indicated by dashed lines (n = 5, mean ± standard deviation)
Table 3.2 Fluorophotometry values for retinal,
mid-vitreous, and cornea following an IV injection
of sodium fluorescein in the anesthetized rabbit
Retina Vitreous Cornea
Pre-IV 6.7853 0.62481
0.5 min 1,632.2 1.105 2.8163
1
min 1,909.3 2.5392 3.1615
2 min 1,137.9 1.4776 2.0636
4 min 928.01 0.9389 12.861
6 min 1,113 1.8254 14.843
7 min 1,467.1 2.3547 18.437
9 min 843.82 0.54 38.876
4.5891
3.8 Ocular Uptake of Fluorescein from Topical Eye Drops
The possibility of delivering a drug to the posterior segment of the eye was evaluated with topical drops of fluorescein applied to a human subject’s eye. Six drops of
10 mL of 7% sodium fluorescein at 15-min intervals was applied. Hourly scans after
last fluorescein application was recorded with the fluorophotometer. The contralateral eye did not receive topical applications of fluorescein. The autofluorescence
values for the ocular tissue of the treated eye (Fig. 3.22a) and contralateral eye
(Fig. 3.22b) show low retinal and corneal fluorescence and a normal high lens fluorescence relative to the 57-year-old subject. Seventy-five minutes after the topical
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