(A) histochemical study of cholinesterase activity in rabbit's retinae
Since early in the 19th century, much research on the relation between acetylcholine and nervous stimulation has been reported. Loewi(1921) reported that a stimulation factor in vagal transmission was found at the synapses and which was similar to acetylcholine in its physiological activity. Frank(1923) indicated that in all synapses of the autonomic systems, the mediator stimulant involving acetycholine was closely related with the nervous conduction. Brinkman and Van Dam(1922) found that following and experimental stimulation of the vagus nerves
induced to secrete acetylcholine which appeared to be included in the irrigated fluid.
Engelhart(1931) noted that, when pupil narrowing occurred with strong light, considerable amounts of acetylcholine-like substance appeared in the aqueous humor of the eye chambers. Dale(1934) confirmed the fact that the activities of acetylcholine was suppressed by esterases, hydropyzing enzymes of the ester. The existence of the high cholinesterase activity in the nerve synapses of skeletal muscles in amphibia, reptiles, and mammals has been biochemically demonstrated by Marnary and Nachmansohn(1983). They explained the reason why these skeletal muscles having motor end plated or other nerve endings should show more strong cholinesterase activity than skeletal muscles without the nerve endings.
Since Gomori(1948) introduced a new method for detection of specific cholinesterase from nonspecific cholinesterase in the various sites(e.g., sympathetic ganglion, the brain). kolle and Friedenwald(1949) designed a good method for detection of cholinesterase using acetylthiocholine iodide or but yrylthiocholine iodide as a substrate in which the hydrolytic activities of specific or nonspecific cholinesterase in the brain, and autonomic ganglia could occur rapidly. Additionally Koelle(1955) improved more reliable histochemical method for acetylcholinesterase.
Recently, much improvements about acetylcholinesterase in mammalian tissues were accompanied, especially those in the nervous system were notable. Hashimoto et al.(1963) reported that cholinesterase activities were demonstrated in the motor end plates, a number of sensory nerves and receptors, various noncornified circumferences of the capillaries, and nerve fiber plexus of the derma, sebaceous glands, and layers of the skin.
A mentioned above, a number of reports on the distribution of cholinesterase were made with subjects mainly related to the nervous and muscular system, and the skin of vertebrates. Moreover, a few reports on cholinesterase activities of the retinae of the eye were found. Koelle and Friedenwald(1950) reported that high cholinesterase cholinesterase activities appeared conspicuously in the inner and outer plexiform layers, and adjacent to the inner nuclear (or granular) layer in rats.
Pak and Choi(1964) indicated that there was a distinct difference of cholinesterase activity between neonatal rabbit's retinae whose palpebrae were still fused and young rabbit's which were able to see. On the other hand, the changes of cholinesterase activity of various organs asociated with the development of the mammalian fetus have been observed by several workers. Kupfer and Koelle(1951) compared the cholinesterase activity of forelimb muscles in rat fetuses with that in neonatal ones. Ehbel(1961) studied cholinesterase activity by tissue culture of the skeletal muscle cells of the chick fetuses. Hashimoto et al.(1963) compared and discussed cholinesterase activities demonstrated in various structure of the fetal skin with the adult ones.
However, a number of histochemical works on cholinesterase activities of various organs and retinae of the vertebrate are reported but also practically nothing on functional relations between the retinae and cholinesterase is to be found.
The present study attempts to observe 1) the normal localities of cholinesterase activity in normal retinae of the rabbit's fetuses and neonatals, 2) the gradual enzymatic changes in the light-isolated animals, and 3) the gradual recovering status of the enzymatic activity in the reopened eyes.
Ⅱ. Materials and Methods
65 healthy young rabbits weighing 300 to 40gm, 33 rabbits' fetuses, and 5 neonatal rabbits with closed eyes were used in this study. In order to observe cholinesterase activity of the retinae in fetuses and neonatals, the rabbits fetuses(15th, 17th, and 20th day of fetal age) obtained from the pregnant rabbits by means of cesarian section, and neonatals were used respectively. In order to observe the gradual changes in the light-isolated ones, young rabbit's palperbrae (the upper and lower palpebrae) were bilaterally sutured by means of the continuous mattress suture, and then were kept in the dark for 1,2,4 and 8 weeks respectively.
In order to observe the gradual recovering status, some of the above animals eyes were reopened by pulling out the silk, and then they were kept in a bright place for 3,7, and 14 days respectively.
The animals were sacrificed by means of the intravenous injection of air. Immediately the enucleasted eyeballs were opened. The excised retinae were fixed in cold formalin-sucrose-ammonia fluid (Person, 1963) for 24 hours and after rinsing in distilled water briefly, the retinal tissues were incubated at 37。C in the substrate containing acetylthiocholine iodide for 2 hours as recommended by Gerebtzoff(1953). Some of the material from the control group was incubated for 6 hours or 12 hours. After treatment of the pieces with 1% ammonium sulfide solution
for 1 to 3 minutes, the retinal tissues were embedded in paraffin by the method of Koelle and Frieden wald(1950), and sectioned at 5 μ.
In order to differentiated the specificity of the diisopropylfuorophosphate (DEP), and irreversible cholinesterase inhibitor for 30 minutes before incubation. The typical dark brown pigmented substances to be proved cholinesterase-sites were
not observed in the cases treated with 10-**3 M solution of DEP and with 10-**5M solution of eserine. The positive reaction of the cholinesterase of the retinae treated with 10-**6M solution of EFP was considerable as the specific cholinesterase.
Some of the preparations were stained with hematoxylin for counterstain. All of the specimens were observed with the light microscope. In order to compare the convenience the retina was divided into three areas; the posterior polar, the equatorial and the peripheral areas.
A.Cholinesterase activity in fetal and neonatal retinae of rabbits.
1. The 15-day fetus group:
It was clear that the retinal structures were not fully differentiated within the 15-day fetuses. Incidentally, specific and nonspecific chilinesterase activities were not were not observed in this group. (Fig.9).
2. The 17-day fetus group:
The histological structures of the retinae in the group were differentiated more than the formor group. However, the activities of these enzymes were relatively similar to the former group.
3. The 20-days fetus group:
The retinal structure was fairly differentiated; the ganglion cell layer and the inner plexiform layer of the retinae began to show slight cholinesterase activities.
4. The neonatal group:
Although the neonatal rabbit's retinae fo the closed eyes could not exhibit their roles in the visual function, after 2 hours of incubation the never fiber layer of the retina showed a weak enzymatic activity and the ganglion cell layer a moderate one. The other retinal layer hardly showed the activities ever in cases of prolonged times for incubation.
B. Cholinesterase activity in the young rabbits.
In the specific cholinesterase activity, the peripheral area near the ora revealed a weak enzymatic reaction in general. However, the ganglion cell layer and the inner plexiform layer showed a weak and distinct reaction. In the equatorial area near the equatorial portion, the ganglion cell, inner plexiform, and inner nuclear layers showed marked activities. In the ganglion cell layer of the posterior polar area, a considerable enzymatic activity was observed compared with the other layers. The cytoplasm of the ganglion cells of this posterior polar area
showed the most strong activity, which was probably in the intracellular site, among those of the other retinal cells. Considerable specific cholinesterase activities were observed not only on the inside of the inner plexiform layer and the inner nuclear layer but also in the outer plexiform layer. In the activity of the nonspercific cholinesterase throughout the whole three areas in this group was faintly positive.
2. The 1 week group after suturing the eyelids:
In the peripheral area of the retina, the inner plexiform layer showed moderate enzymatic activity of the specific cholinesterase, and the ganglion cell layer slight activity. In the equatorial area, the ganglion cell layer was more marked than that of the peripheral one. In the posterior polar area, the ganglion cell layer was the most remarkable in activity. Comparing with the control group, the enzymatic activities in this group were less than these control group. A wide distribution pattern of the enzymatic activity even to outer plexiform layer was also in this group as in the control group.
3. The 2 weeks group after suturing the eyelids:
The specific cholinesterase activities of the retinal layers were fairly reduced compared with the 1 week group. But the three retinal layers were clearly distinguishable in all of the preparation.
4. The 4 weeks group after suturing the eyelids:
All of the retinal layers were distinguishable but the enzymatic activities of all the layers were markedly reduced. Only several ganglion cells showed a weak enzymatic reaction.
5. The 8 weeks group after suturing the eyelids:
All of the retinal layers were still distinguishable. Enzymatic activities in this group were similar to those of the former group. In the posterior polar ares, the ganglion cell layer and the inside of the inner plexiform layer showed slight activity. Additionally nonspecific cholinesterase in these experimental group showed less significance throughout the whole retinal areas.
6. The 3 days group after reopenning eyes:
In the peripheral area of the retina, the specific cholinesterase activity of the ganglion cell layer began to sho recovery of the enzymatic activity and the outside of the inner nuclear layer showed weak activity. In the equatorial area, the
activities of the ganglion cell layer and the inner unclear layer were less than those of the peripheral area. In the posterior polar area, the ganglion cell layer showed more enzymatic activity than that of the outer retinal areas.
7. The 7 days group after reopenning eyes:
In the retinal areas and layers, the cholinesterase activities had recovered and were similar to those of the control group.
8. The 14 days group after reopenning eyes:
In the retinal areas and layers, the enzymatic activities were almost recovered and observed as seen in the normal group.
Ⅳ.Summary and Conclusion
In the present study the specific and nonspecific cholinesterase activities of the rabbit's retinae in the fetus, the neonatal, the normal, the light-isolated, and the reopeneds groups were histochemically observed.
1. Cholinesterase activity of the retinae in the 15 days fetuses were not found but began to show in the 20 days fetuses.
2. In the 1 week group after suturing the eyelids, the most remarkable activity of specific and nonspecific cholinesterase was observed in the posterior polar area among the retinal areas. The nearer to the peripheral area the weaker enzymatic activities were observed. In the 2 weeks group after suturing eyelids, the
enzymatic activity were reduced. In the 4 and 8 weeks groups after suturing eyelids, the enzymatic activities were remarkably reduced.
3. In the 3 and 7 days groups after reopenning eyelids, both enzymatic activities were considerably recovered, especially remarkable in the peripheral area of the retina. In the 14 days group after reopenning eyelids, enzymatic activities were almost similar with the normal control group.
Consequently it is histochemically deduced that the gradual changes of specific cholinesterase activities in the rabbit's retinae were closely related to the visual function.