시신경 절단이 가토 망막의 Cholinesterase 및 Acid phosphatase 활성반응에 미치는 조직화학적 연구

Abstract

[한글]

[영문]

Ⅰ. Introduction

Following the report of Loewi(1921) that a stimulating factor for vagal transmission was located at the synapse and that this factor was similar to acetylcholine in its physiological activity, Dale(1934) proposed that acetylcholine transmitted impulses at the neuromuscular junction.

Gerebtzoff(1955) postulated that transmission of neural impulses in the retinal synapses was probably associated with the acetylcholine-cholinesterase system. Anfinsen(1944) found high cholinesterase activity at the neural synaptic junction in the retina of the ox and strongly proved Dale's theory that acetylcholine is the chemical transmittor of neural impulses at the synapses. In addition, Koelle and Friedenwald(1950), studying the rat's retina, reported that high cholinesterase activity was conspicuously seen in the inner and outer plexiform layers as well as adjacent to the inner nuclear layer.

With regard to the visual function associated with retinal cholinesterase activity in the experimental animal, Liberman(1962) found that the specific cholinesterase activity of the retina of the control young dog was greater than that found in the retina of the experimental dog which had been bred and lived in the dark since birth. Only a few histochemical studies of cholinesterase activity in animal retinae with emphasis on visual function have been reported.

The present study observes 1) the histochemical changes in cholinesterase activity in rabbit's retinae, 2) the histochemical features of acid phosphatase activity in the retinae, and 3) the histochemical alteration of neuronic pyroninophilic granules and histological structures of the retinae following severance of the optic nerve.

Ⅱ. Methods and Materials

55 healthy young rabbits weighing 300 to 500gm were used in this study. Under ether anesthesia horizontal incision was made in the skin of the upper right palpebra. The upper bony part of the orbital margin was removed to approach the posterior pole of the eye ball by widening the surgical field. The optic nerve was

severed 3 to 4mm from the site where the optic nerve pierces the sclera. Vascular injury of the blood vessels which enter the eye ball through the posterior pole was avoided. Also posteoperative retinoscopy was done to eliminate unsuitable animals as well as preoperative one. The intact left retina of each animal was used as a control.

Following weverance of the optic nerve, 15 rabbits were fed 1 week, 15 rabbits for 2 weeks, 13 rabbits for 4 weeks, and 12 rabbits for 8 weeks. Then each rabbit was sacrificed by injection of air through the ear veins.

1. Histochemical demonstration of cholinesterase activity:

Excised retinae were fixed in cold formalin-sucrose-ammonia fluid (Pearson, 1963) for 24 hours. After being rinsed briefly in distilled water, frozen sections, 10 microns in thickness, were made. The section was incubated for 2 hours at 37 degrees C. in the substrate containing acetylthiocholine iodide (Gerebtzoff, 1953) and mounted in histoclad. To differentiate the specificity of cholinesterase activity, a 10**-6 M solution of diisopropylfluorophosphate (DFP) was used as a preincubating medium for specific cholinesterase. A 10**-3 M solution of DFP, or 10**-5 M solution of eserine, was used for total inhibition of specific and nonspecific cholinesterase.

2. Histochemical method of acid phosphatase cholinesterase.

The excised retinae were fixed in cold neutral formol calcium solution for 24 hours. Frozen sections, about 10 microns in thickness, were prepared.

The retinal section was incubated for 20 minutes at 37 degrees C. in Gomori's (1941) medium as modified by Eranko(1952). This contains sodium beta-glycerophosphate as a main substrate. Sections were then mounted in histoclad. For a control testing of acid phosphatase activity, the section was similary incubated in a medium lacking the main substrate of sodium beta-glycerophosphate. These controls all showed absence of acid phosphatase activity.

To demonstrate pyroninophilic granules in the retinal cells histochemically and show the histological structures of the retina as a whole, Rosa's (1950) method of methyl green-pyronin was used.

Ⅲ. Results

A. Cholinesterase activity in the young rabbit's retinae.

1. Specific cholinesterase activity in the control retina (left) of the rabbit's eye one week after severance of the right optic nerve:

In the intact left retina specific cholinesterase activity was demonstrated in the cytoplasm of the ganglion cells (ganglion cell layer) and in the inner plexiform layer. Also light enzymatic activity was frequently observed in the inner nuclear and outer plexiform layers.

2. Specific cholinesterase activity in the experimental retina (right) of the rabbit's eye one week after severance of the right optic nerve:

The right retinae showed enzymatic activity of specific cholinesterase in the ganglion cell and inner plexiform layers which was comparable to that of the preceding group. Thus the specific cholinesterase activity, within the one week period, was not significantly altered by severance of the optic nerve.

3. Specific cholinesterase activity in the control retina (left) of the rabbit whose right optic nerve had been severed 2 weeks previously:

In this control retina specific cholinesterase activity was similar to that in the control retina of the preceding control group. Enzymatic activity of the left retina was not affected 2 weeks after severance of the right optic verve.

4. Specific cholinesterase activity in the experimental retina (right) of the rabbit whose right optic nerve was severed 2 weeks previously:

In the retina there was less specific cholinesterase activity in the cytoplasm of the ganglion cell than in the control left retina. However, the enzymatic activity showed little change in the inner plexiform and other retinal layers.

5. Specific cholinesterase activity in the control retina (left) of the rabbit's eye whose right optic nerve had been severed 4 weeks previously:

In this left retina specific cholinesterase activity was similar to that of the control retina of the preceding group. Enzymatic activity in the retina underwent very little alteration in this 4 weeks experimental period.

6. Specific cholinesterase activity in the experimental retina (right) of the rabbit whose right optic nerve had been severed 4 weeks previously:

At this time the enzymatic activity of specific cholinesterase could be demonstrated in each layer of the retina as in the previously group. However, its activity in the neural cytoplasm of the ganglion cell (ganglion cell layer) and the inner plexiform was reduced. This enzymatic alteration clearly demonstrates a decline in enzymatic activity in the experimental retina (right) 4 weeks after optic nerve transection.

7. Specific cholinesterase activity in the control retina (left) of the rabbit whose right optic nerve had been severed 8 weeks previously:

In this control retina (left) specific cholinesterase activity was similar to that of the control retina of each of the previous groups.

8. Specific cholinesterase activity in the experimental retina (right) of the rabbit whose right optic nerve had been severed 8 weeks previously:

Enzymatic activity of specific cholinesterase was only slightly demonstrable in the neural cytoplasm of the ganglion cell and in the inner plexiform layer.

In summary: Eight weeks after severance of the optic nerve and cutting the connection to the central nervous system in the rabbit there was a fairly demonstrable decrease in the ipsilateral retinal specific cholinesterase activity.

B. Acid phosphatase activity in the young rabbit's retinae

1. Acid phosphatase activity in the control retina (left) of the rabbit whose right optic nerve had been severed 1 week previously:

In the left side of the control retina small granular activity of acid phosphatase was observed in the perikaryon of the ganglion cell (in the ganglion cell layer) and in the neural cytoplasem of the inner nuclear layer.

2. Acid phosphatase activity in the experimental retina (right) of the rabbit whose right optic nerve had been severed 1 week proviously:

In the experimental retina (right) the enzymatic activity of acid phosphatase was fairly similar to that of the control as above.

3. Acid phosphatase activity in control retina (left) of the rabbit whose right optic nerve had been severed 2 weeks previously:

Enzymatic activity of acid phosphatase was similar to that of the control retina in the previous group.

4. Acid phosphatase activity in the experimental retina (right) of the rabbit whose right optic nerve had severed 2 weeks previously:

The enzymatic activity of acid phosphatase was generally increased in the perikaryon of the ganglion cell and in the neural cytoplasm of the inner nuclear layer. Also small granules, probably contained in lysosome, which had been demonstrated previously in the positive reaction of acid phosphatase, had enlarged (and converted to probable cytolysome).

This result may be comparable with the acid phosphatase activity observed in the neuron, which undergoes degeneration.

5. Acid phosphatase activity in the control retina (left) of the rabbit whose right optic nerve had been severed 4 weeks previously:

The enzymatic activity was generally similar to that of the control retina of the preceding group. However, enzymatic activity as stronger in the neural cytoplasm of the inner nuclear layer than that noted in the control retina.

6. Acid phosphatase activity in the experimental retina (right) of the rabbit whose right optic nerve had been severed 4 weeks previously:

The enzymatic activity in the neural cytoplasm of the ganglion cel layer and the inner nuclear layer was remarkbly decreased. The decline in enzymatic activity seen so soon after severance of the optic nerve is comparable to the changes in enzymatic activity in other neurons which have undergone a Wallerian degeneration.

7. Acid phosphatase activity in the control retina (left) of the rabbit whose right optic nerve had been severed 8 weeks previously:

Enzymatic activity was similar to that of the other controls.

8. Acid phosphatase activity in the experimental retina (right) of the rabbit whose right optic nerve had been severed 8 weeks previously:

This experimental retina showed a marked decline of enzymatic activity in the neural cytoplasm of the ganglion cell and inner nuclear layer as compared with the 4 weeks experimental group.

In summary, the enzymatic activity of acid phosphatase in the retina after severance of the optic nerve once increased and then decreased. This phenomenon was similar to that seen in other neurons which have undergone a Wallerian degeneration. However, the decline of acid phosphatase activity in the neural cytoplasm of the ganglion cell layer and the inner nuclear layer came earlier than has been reported by others.

C. Pyroninophilic granules in the retina of the young rabbit.

There are a moderate number of pyroninophilic granules in the cytoplasm of the ganglion cell and inner nuclear layers. 4 weeks after severance of the optic nerve, the granules in the cytoplasm of the ganglion cell had decreased. There are less of a decrease in the granules in the cytoplasm of the inner nuclear layer.

Ⅳ. Summary and Conclusion

Young rabbits, whose right optic nerves had been servered in the orbit, were fed for 1 week, 2 weeks, 4 weeks and 8 weeks respectively. The retina of the left eye was as a control and that of the right eye as an experiment. Using histochemical methods the cholinesterase, acid phosphatase and ribonucleic acid and the histochemical changes of the retina due to severance of the optic nerve were observed for 8 weeks after section.

1. In the young rabbit's retina, whose visual connection to the central nervous system was blocked, specific cholinesterase activity was decreased beginning at the 4th week after section of the nerve. At the 8th week. the enzymatic activity in the perikaryon of the ganglion cell and the inner plexiform layer was only weakly demonstrated.

2. Acid phosphatase activity in the young rabbit's retina was increased at the 2nd week, but was decreased below normal from the 4th week. This rapid decline of acid phosphatase activity was characteristic in the experimental retina and was comparable to the rather slow alteration of enzymatic activity in neuron undergoing a Wallerian degeneration.

3. Pyroninophilic granules contained in neural cytoplasm of the retina were affected by surgical block of visual connection with the central nervous system. The granules had partly disappeared from the perikaryon of the ganglion cell and from the inner layer beginning at the 4th week.

Consequently, as the results of histochemical studies, it is postulated that first the gradual decline of specific cholinesterase activity in the rabbit's retina was closely related to an intraorbital block of the optic nerve, secondly

that the typical degeneration of the ganglion cell in the ganglion cell layer, which was associated with a partial disappearance of the ganglion cellm was related to the changes in acid phosphatase activity and alteration of the pyroninophilic granules in the retina following optic nerve optic nerve transection.