After injection of fluorescent 70-kDa dextran into the anterior chamber of NIH Swiss mice, fluorescence was observed sequentially in tissue compartments that typically comprise the trabecular meshwork and uveoscleral outflow pathways. First, the tracer was visible in the trabecular meshwork and ciliary process stroma. Next, it was observed in the ciliary muscle and anterior choroid. Later, it was observed in equatorial choroid and within the stroma of adjacent equatorial sclera. Finally, there was a reduction of the fluorescent signal present in most of these structures, with retention of fluorescence observed only in the trabecular meshwork and Schlemm’s canal. Thus, in these latter two structures, there may have been binding or endocytosis of the fluorescent dextran. In contrast, the gradual elimination of fluorescence within the elements of the uveoscleral outflow pathway, indicates that there was minimal binding of the labeled dextran to these tissues. Hence, the progression of fluorescent signal through uveoscleral outflow pathway in the mouse probably reflects bulk transport of the labeled dextran as part of normal aqueous humor drainage from the anterior chamber.
The sequence of tissues in which the fluorescent tracer appeared in the present study is the same as in monkey eyes in which uveoscleral outflow has been well characterized. In an early study, intracamerally injected anionic ferritin distributed through the extracellular spaces of the monkey ciliary muscle and choroid within 20 minutes.
6 The tissue distributions of intracamerally injected vinyl particles also confirmed bulk aqueous humor movement from the monkey anterior chamber through the ciliary muscle to choroid.
9 Additional studies identified the bulk movement of aqueous humor in the monkey uveoscleral outflow pathway from intraocular tissues to the sclera, and then to extraorbital tissues.
10 21 22 Similar labeling of the ciliary muscle, choroid, and sclera were obtained in a study of labeled albumin movements after intracameral injection into human eyes.
23 The appropriateness of using fluorescent dextran as a marker for bulk flow in the mouse eye is supported by previous studies showing that fluorescent dextrans are stable in vivo and in vitro
24 and that they are suitable for characterizing bulk flow of aqueous humor through the uveoscleral outflow pathway in monkey.
5 25 Thus, the movements of fluorescent dextran in the mouse eye observed in the present study suggests a uveoscleral outflow pathway exists in the mouse that shares many features of the uveoscleral outflow pathway of the monkey.
Although there are striking similarities in the anatomic features of the uveoscleral outflow pathways of the mouse and monkey, there also may be differences between them. For example, the thin sclera of the mouse eye may be more permeable to transscleral movement of molecules from the suprachoroidal space to the extraorbital tissue space. This is suggested by the absence of observed fluorescence in the mouse posterior pole choroid compared with the appearance of tracer in the monkey posterior pole choroid.
3 It is possible that the amount of tracer injected was too low to deliver sufficiently observable label to the posterior pole. Another possibility is that the size of the dextran tracer may be important. Previously, Toris et al.
5 examined the ocular tissue distributions of 4-kDa, 40-kDa, and 150-kDa dextrans after intracameral injection in normal and inflamed monkey eyes. They observed good recovery of 40-kDa dextran in anterior uvea, anterior sclera, and posterior sclera of normal eyes. However, the most efficient recovery in the posterior sclera of inflamed eyes was with 150-kDa dextran.
5 Continuity of the posterior pole and equatorial choroidal spaces in mouse is suggested by the similar labeling of these compartments after subconjunctival injections of 70-kDa dextran.
26 Thus, additional experiments with higher dose injections of 70-kDa dextran, as well as with higher-molecular-weight dextran tracers, may further clarify the properties of macromolecular transport to the posterior pole of the mouse eye by uveoscleral outflow.
In addition to the observed fluorescence in the uveoscleral outflow pathway, fluorescence also was observed in the trabecular meshwork and Schlemm’s canal. This observation is consistent with an important role for trabecular meshwork outflow in the normal drainage of aqueous from the mouse anterior chamber. It is likely that despite delivering the injected dextran over the course of several minutes, there was elevation of IOP associated with the injection. Because conventional outflow is largely pressure sensitive, it may have increased conventional outflow of the tracer beyond normal in the present studies.
7 13 However, it is likely to have had little influence on the movement of tracer in the present study, because uveoscleral outflow facility appears to minimally affected by increased IOP in monkey eyes.
7 13 Physiological studies, as have been conducted in monkeys and other species,
1 14 27 28 29 30 are needed in the mouse eye to investigate these questions and to determine the proportion of total outflow that passes through the trabecular meshwork route versus the uveoscleral outflow pathway. Previous studies showing that fluorescent dextran can be used to measure aqueous humor dynamics in the eyes of cats and rabbits suggest that it also may be useful to measure outflow dynamics in mouse eyes.
31 32
In conclusion, the present study provides direct evidence supporting the presence of a mouse uveoscleral outflow pathway that has many similarities with the monkey uveoscleral outflow pathway. In particular, analogous structures within the uveoscleral outflow pathway appear to be involved in both the mouse and primates. These results, coupled with the prior observations of IOP reduction in the mouse eye after topical instillation of the PG analogue latanoprost,
17 18 raise the possibility that the mouse eye may be a useful model system in which to investigate general mechanisms of uveoscleral outflow regulation. Many mutant and transgenic mice strains are available, including some in which abnormally elevated IOP has been identified.
33 34 35 36 Thus, the mouse eye may provide new opportunities for studying the cellular and molecular mechanisms that influence uveoscleral outflow.
The authors thank Makoto Aihara, MD, PhD, for assistance in the development of the microinjection methods use in the study.