- Email: [email protected]
The use of Isolated Retinal Tissue in Studies of the Metabolism of the Central Nervous System*
T H E USE O F ISOLATED R E T I N A L T I S S U E IN STUDIES O F T H E METABOLISM O F T H E CENTRAL NERVOUS SYSTEM* W.
A. ROBBIE, P H . D . , AND P. J. LEINFELDER,
M.D.
Iowa City, Iowa
Cellular respiration processes are extremely important for both the functioning and survival of the cerebral cortex and the retina and, if the supply of either oxygen or substrate is interrupted, unconsciousness follows quickly. Manometric studies on the metabolism of isolated brain have revealed something of the nature of the enzyme systems that are involved in oxidation, and of the effects of anesthetics and other pharmacologie agents. A comparison of the metabolism of the cerebral cortex and the retina may be of interest to the ophthalmologist because of interest both in the retina itself and in the information that retinal studies may give about the physiology of the central nervous system. Physiologically, the retina resembles cerebral cortex in its high rate of oxygen consumption, its unusually high rate of both aerobic and anaerobic glycolysis,1 its dependence upon glucose substrate, and the almost complete depression of respiration produced by heavy metal inhibitors.2 Since the retina is derived from neuroectoderm, a morphologic similarity is to be expected, and this has been shown to be true up to the 45-mm. stage of embryologie development.3 In the adult tissues, in spite of specialization of each, a similarity remains in the lamellar arrangement of nerve cells, synaptic, and nerve-fiber layers. Isolated rat retina is a convenient tissue for use in manometric metabolism measurements. It is easily and quickly removed from the eye with relatively slight injury, and is thin enough to permit adequate oxygιnation without slicing, even though air is the gas phase rather than 100-percent oxygen. * From the Departments of Ophthalmology and Physiology, College of Medicine, State University of Iowa. Aided by a grant from the John and Mary R. Markle Foundation.
If the suspension medium is buffered and contains glucose and a properly balanced salt mixture, linear respiration may be obtained for a period of 5 to 7 hours. It is thus possible and convenient to measure respiration during a control, experimental, and recovery period on the same tissue. Since the rat retina does not break up while it is shaking in the manometer, it may be easily removed and weighed at the end of the experiment. Experimental determination of respiration in isolated brain is complicated by the procedure of cutting thin slices of the tissue in order to insure adequate oxygιnation. Since the nerve cells are extensively branched, it is evident that a slice of optimum thinness must have a high proportion of cells with cut processes. It is recognized that cutting a nerve process may cause degeneration of the cell body even though the cell is otherwise undisturbed. In an artificially prepared saline solution, there may be additional injury from diffusion into the cell of ions in a concentration different from the normal constitution. Certain experimental agents that do not penetrate the intact cell may likewise adversely and misleadingly affect the brain slice. The necessity for use of 100-percent oxygen also complicates certain determinations because of the oxygen-poisoning effect. TECHNIQUE
The experiments described in the present paper were performed on albino rats. The animals were killed by breaking the neck, and either the eyes or the brain was removed immediately. In studies on cerebral cortex, the brain was sliced by means of a razor blade and a transparent plastic template.4 The slices were weighed on a microtorsion balance before they were immersed in physi-
208
RETINA IN METABOLISM STUDIES ologic saline solution. Retinas were removed by cutting around the globe just posterior to the ora serrata. The retinas were then lifted out and cut into halves to prevent folding. The isolated tissues were suspended in phosphate-buffered saline solution at pH 7.35 containing 0.2-percent glucose.5 Small Warburg manometer flasks containing a total volume of about 7 cc. were used for the oxygen consumption determinations. The center wells contained 10-percent KOH on filter paper. Temperature of the water bath was 37.2°C. Unless otherwise mentioned, experiments on retinas were performed with air as the gas phase and those on brain slices with 100-percent oxygen. Retinal weights were determined after rinsing the tissues at the end of the experimental periods and drying them on weighed cover glasses at 110°C. The dry weights for the brain slices were calculated from the recorded wet weights by means of a wetdry ratio obtained by drying sample tissue at 110°C. . In the histologie studies, brain tissue was prepared in the following manner: For the control sections, Figure 5 (left), the brain was removed immediately after the death of the animal, and slices of the cerebral cortex were cut and placed in 10-percent neutral formalin for fixation. In other specimens studied, slices of tissue were placed in manometer flasks in the phosphate-glucose-saline solution. These flasks were then placed in the water bath and the respiration rate was recorded for one-half and 2j4-hour intervals in order to verify the viability of the tissue. Following this, the tissue was removed from the flask and fixed. A similar procedure was used in experiments on the rat retina. In addition, tissue slices and blocks from rat brain were stored in saline solution at 4°C. for intervals varying from 2 to 24 hours. At the end of the storage period, the brain tissue was placed in formalin for fixa-
209
tion. Sections made from all preparations were stained with hematoxylin and eosin. EXPERIMENTAL RESULTS AND DISCUSSION
Results of a number of experiments seem to indicate that in the tissues studied oxygen consumption is not dependent upon the integrity of the cell. Both cortex slices or chopped tissue made by cutting the cortex into small pieces respire at about the same level. Similarly, either intact retina or retina which has been cut into small pieces shows the same oxygen uptake for a period of at least one hour. However, if the tissues are ground up in a tissue grinder, then the respiration falls off considerably. It is possible that as long as the gross intracellular organization is not appreciably disturbed, the respiratory enzyme systems may function at a normal rate. Figure 1 shows that when a properly buffered and balanced saline solution containing glucose is used, the respiration of the retina in air is maintained at a constant rate for as long as 7 hours. The lower curve in the figure shows the course of oxygen consumption when a retina is run in fluid containing no glucose. The rapid decline in respiration to almost the zero level is convincing evidence that the process being measured in the respirometers is glucose metabolism rather than any sort of autolytic degenerative change. There is a pronounced difference in the course of oxygen consumption of the retina in air and in 100-percent oxygen. In air, the rate of respiration is constant during the observation period. In oxygen, the rate of respiration is higher at the start of the measurement period, but there is a marked falling off and, by the time the experiment has been run for a period of 7 hours, the oxygen consumption has decreased to 50 percent of that of the specimen in air. This is shown in Figure 1. The curve for brain respiration is similar to that for retina in oxygen and it may be
210
W. A. ROBBIE AND P. J. LEINFELDER
that if it were possible to run cortex slices in air, the respiration would be more constant. This is not feasible since a tissue slice that is thin enough to permit adequate oxygιnation at the center, with air as the gas phase, is almost impossible to prepare. The steady rate of respiration of the retina in air perhaps more nearly resembles the situation in the living animal, since the oxygen tension within the eye can certainly
0
tex slices, the response of the injured cells to certain chemical factors may be considerably different than would be expected from normal tissue. It is well known that highly dissociated acids do not go through the normal cell membrane. Figure 2 shows the results of an experiment with brain slices and retinas in which the tissues were exposed to a saline solution at pH 5.2 for a period of one hour, after
Z
A-
6
HOURS Fig. 1 (Robbie and Leinfelder). Oxygen consumption of isolated rat retinas in phosphate-buffered saline solution containing 0.2 percent glucose. Ordinate shows the mm.3 of 0 2 consumed per mg. dry weight, and the abscissa the time in hours.
never approach that which exists in the flasks gassed with .100-percent oxygen. Whether the increase in oxygen consumption of the retina in oxygen at the start of the measurement period is actual respiration or simply oxidation of substrates that might not otherwise be utilized, is a question. The falling· off in respiration at the end of the 6- or 7-hour period of measurement may possibly be an oxygen-poisoning effect. Although the rate of respiration is perhaps not immediately affected by cutting the processes of the nerve cells in cerebral cor-
which the recovery oxygen consumption was measured. Although the retina has apparently not been damaged by this exposure to an acid environment, the cortex slices with their· cut cells and processes show considerable injury, as evidenced by a marked decline in respiration. Kidney and liver slices also have been shown to recover almost completely after an hour's exposure to a medium at pH 5.6 Figure 3 shows the results of an experiment using 0.1 M potassium chloride in the suspension fluid. In vivo, the potassium ion
211
RETINA IN METABOLISM STUDIES penetrates the brain very slowly.7 Yet the respiration of the brain slices is increased by almost 100 percent when a high concentration of potassium is included in the saline solution. (The sodium chloride concentration in these experiments was reduced to maintain an isotonic medium.) The retina, on the other hand, shows, if anything, a slight decrease in oxygen consumption in the medium containing high potassium. Similar observations have been reported by Dickens.5 This again indicates that potassium, whatever may be the nature of its stimulating action, apparently does get into the cortex cells which have been cut, but not into the retina cells. The cytologie picture also gives evidence that the brain is much more injured during
With brain tissue, on the other hand (fig. 5), edema was noted after one-half hour in the Warburg flasks, and the ganglion cells had begun to undergo chromatolytic change, as shown by the swelling of the
16
o
«4-
A
/
«V / y
30
o ιο
0
*.·
*?/
éf
/
^Ã
*w
# #
1
/
y
M" Z
"
0 HOURS
1
I
40
O MINUTES
40
Fig. 3 (Robbie and Leinfelder). Effect of addition of 0.1M. KC1 to phosphate-buffered saline solution on the respiration of retina and cerebral cortex slice. Ordinate : mm." of Oδ per mg. dry weight.
BRAIN
RETINA
.-'
<
20
40·
BRAIN
«TINA
<<***..
1
2
Fig. 2 (Robbie and Leinfelder). Recovery of retinas and cerebral cortex slices after one hour at pH 5.2. During the exposure period the tissues were shaken in manometer flasks in glucose-saline solution containing 0.01M. primary sodium phosphate. Ordinate shows oxygen uptake during recovery period in mm." Oj per mg. dry weight. preparation for respiration measurement than the retina. Figure 4 shows sections from a control retina and one which had been run for 2% hours in a manometer flask at 37.2° C. The ganglion cells and the other retinal cellular elements were similar in both the control and in the experimental tissues at the end of the experiment.
cytoplasm and nuclei. In some instances, the ganglion cell nuclei were extremely vacuolated, indicating karyorrhexis. These changes were considerably exaggerated in a 2J^-hour specimen and many of the ganglion cells had disappeared, while all of those remaining showed advanced chromatolysis. In some instances, glial clumping about the ganglion cells could be observed. A similar type of change was observed after two hours' storage at 4°C, and this became increasingly apparent after longer storage periods. When the brain remains intact in the dead animal at room temperature for as long as ΐÕæ hours, chromatolytic changes do not occur. However, if the brain is removed and tissue slices or blocks are prepared, evidence of cellular damage can be recognized within half an hour if the tissue is maintained at 37° C. ; similar changes are apparent after two hours when the tissue is kept at 4°C. After greater periods of time, more severe
212
W. A. ROBBIE AND P. J. LEINFELDER
Fig. 4 (Robbie and Leinfelder). Photomicrographs of sections of rat retinas. (Left) Control section. (Right) Section of retina shaken for 2 ^ hours in manometer flask at 37.2° C.
Fig. 5 (Robbie and Leinfelder). Sections of rat cerebral cortex slices. Control on left fixed immediately after death. Experimental tissue on right shaken for one-half hour in manometer flask before fixation.
RETINA IN METABOLISM STUDIES
chromatolytic phenomena are apparent. These changes, which are identical with those occurring in encephalomalacia, appear to be the result of injury to the ganglion cells or their processes, and the chromatolytic response may occur directly as a result of the injury to the cells or indirectly because of the change in the intracellular chemical environment that occurs when the cut cells are placed in physiologic saline solution. (The concentrations of certain ions in protoplasm are quite different than those in blood plasma.) In the preparation of retinal tissue, the ganglion cells themselves are not directly injured and only the axone is cut. This injury to the retinal ganglion cell axone occurs at some distance from the cell body, and is less severe trauma than that which occurs to the ganglion cells of the brain when tissue slices are prepared, for, in the latter case, both cell processes and the cells themselves are cut. With the brain slices, a much greater proportion of the cell cytoplasm is exposed to an abnormal chemical environment than in the retina when the main mass of the tissue has not been disrupted. CONCLUSIONS
The structural and physiologic similarity between the cerebral cortex and the retina
213
make it seem probable that the respiratory response of the retina is, in many ways, truly indicative of central nervous system metabolism. Rat retinas are convenient to prepare and to use in manometric determinations, and the period of constant oxygen uptake is long enough to allow measurement of experimental treatment periods and recovery in the same tissue. Injury to the cell membrane in cerebral cortex slices is evidenced by hydrogen ion effect and potassium stimulation of respiration, as well as by a rapid cytologie response to the injury. In studies involving an agent that normally does not penetrate the brain cell, results may be more representative if measurements are made on the retina rather than on a brain slice. The toxic action of pure oxygen on the retina, and the similarity of the curves representing respiration in oxygen of the retina and the brain, also indicate that brain metabolism in 100-percent oxygen is probably not normal. It is believed that studies of the retina may in some ways be more clearly indicative of central nervous-system metabolism than observations on brain slices, and it may be advisable to use the retina for comparison and control experiments. University Hospitals.
REFERENCES
1. Warburg, O. : άber die Klassifizierung tierischer Gewebe nach ihrem Stoffwechsel. Biochem. Ztschr., 184:484,1927. 2. Robbie, W. A., and Leinfelder, P. J. : Cyanide inhibition of retinal respiration in bicarbonate buffer. Arch. Biochem., 16:437,1947. 3. Haden, H. C. : Concerning the similarity of the developing retina and brain wall in human embryos. Am. J. Ophth., 28:943, 1945. 4. Fuhrman, F. A., and Field, J., 2d: Action of diphenyloxazolidinedione on brain respiration at varied temperature levels. J. Pharmacol. & Exper. Therap., 77:229,1942. 5. Dickens, F., and Greville, G. D. : CLXXVII. The metabolism of normal and tumour tissue. XIII. Neutral salt effects. Biochem. J., 29:1468,1935. 6. Stearns, A. W., Jr., Greenblatt, M., Canzanelli, A., and Rapport, D.: The reversibility of pH effects on the ft consumption of tissues. Am. J. Physiol., 132:564,1941. 7. Fenn, W. O. : The role of potassium in physiological processes. Physiol. Rev., 20:377, 1940.
214
W. A. ROBBIE AND P. J. LEINFELDER DISCUSSION
DR. T. D. DUANE (Iowa City, Iowa) : Granit in Sweden, Adrian in England, Bronk, Hartline in the United States have shown that the retina is an ideal tissue upon which to study electrical phenomena in the body, action potential currents, secondary to stimulation, and so forth. Dr. Robbie has just pointed out that the retina is an ideal tissue upon which to study metabolic activities of the central nervous system. It seems to me this is a typical example of the point which Dr. Friedenwald was making last night, that we are opening new vistas in research in ophthalmology, because' the next obvious step is to make a correlation between electrical phenomena and metabolic phenomena. In other words, it seems conceivable to me that one might injure the various portions of the retina, say the rods and cones, bipolars, or the ganglion cells, to see what effect that has upon the electrical phenomena in the electroretinogram, and then the retina could be removed and studied in vitro from the metabolic standpoint and I think some very interesting correlation might be made. DR. P. J. LEINFELDER (Iowa City, Iowa) : The portion of this paper that has been particularly interesting and spectacular to me is the portion dealing with the pathology in the nervous system. Unfortunately, the lantern slides do not show the great degree of chromatolysis that occurs in the ganglion cells of the brain. You who have taken photomicrographs of brain tissue at 800 or 900 magnification know how difficult it is to show representative areas in a particular slide. However, the effects of slicing the brain tissue are real and occur with great rapidity for they can be clearly recognized one-half hour after sectioning. The paradox of the situation is this. If we leave the brain intact in the animal for 2,4,6 hours, and then remove the cal-
varium, take out the brain, take a thin slice of it, and put it in fixative immediately, we obtain perfectly normal histologie appearances. However, if the brain tissue is sliced and then allowed to lie in saline buffer solution with glucose and oxygen available, the changes of chromatolysis take place. Such changes are not recognized in the retina up to 6 hours when the temperature is 37.5°C, nor up to 24 hours when the retinas are kept at 4°C. The occurrence of these chromatolytic changes in nerve tissue which is assumed to be dead is a particularly interesting problem and one which has to be investigated in the light of what is taking place in the tissues as far as respiration, glycolysis, and utilization of substrate is concerned. DR. DAVID G. COGAN (Boston, Massachu-
setts) : I would like to ask Dr. Robbie if there is any contradiction in the apparent susceptibility of the retina to anoxemia, as judged by "black-outs," and the apparent resistance of the retina to oxygen lack as determined by studies in the Warburg apparatus. DR. ROBBIE (closing) : I would like to say that I have no way of knowing what effect the proteolytic enzymes may have on the histologie structure of the tissue. To Dr. Cogan's comment I may say that I think the two factors are possibly quite dissociable —oxygen consumption and the preservation of the visual functions. It is well known of course that the anoxic retina loses its power of perceiving light very rapidly, and anoxia for a period of 7 minutes in the intact eye leads to permanent blindness. Yet if we kill the animals and keep the dead bodies at 37° C. for a period of 45 minutes and then take the retinas out and measure them in the manometer flasks, the oxygen consumption is apparently normal. So it seems as if the limiting element in the system is something other than a chain of respiratory enzymes.
METABOLISM OF THE CRYSTALLINE LENS It may be that the ganglion cells themselves are particularly susceptible in some way morphologically or chemically. To Dr. Duane's contribution, I would like to discourse somewhat on his use of the word "ideal." We do not believe that the retina is necessarily ideal for these purposes. For example, Dr. Friedenwald suggested to me that perhaps the pigment layer is important in retinal function. When we take the retina out of the eye and leave the pigment layer behind, we no longer have a completely normal retina, and perhaps we are disturbing the function in that way. Furthermore, when an animal is killed, unless the eye is fixed immediately, the rod and cone
215
layer quickly becomes diffused in appearance. So the retinas we are studying probably do not have an intact rod and cone layer. What we are measuring is the metabolism of the surviving cells. Any conclusions regarding vision that involve the photochemistry of visual purple are perhaps not shown by these studies. The one point that I do wish to make is that possibly the retina is more representative of the normal central nervous system than the cerebral cortex slice, and at least it may be valuable to compare both types of tissues when the effect of a drug is being studied so that it may be possible to evaluate the factors of permeability and injury.
METABOLISM O F T H E CRYSTALLINE L E N S * I. WATER CONTENT AND GROWTH RATE LAWRENCE O. ELY,
M.D.
Iowa City, Iowa Water is quantitatively the major constituent found in the crystalline lens. Accurate interpretation of certain lenticular properties, namely, Q 0 2 , is dependent upon a precise knowledge of the ratio of wet weight to dry weight. Bellows1 presents a table summarizing the information in the literature concerning the water content of bovine lenses (Table 1 ), but it is of note that the cases cited by any one worker are too few in number to be of statistical significance except for Salit's2 figure of 65.41-percent water for 1- to 4-year-old bovine lenses. However, Krause 3 gives a statistically reliable water content of 67 percent in 1-yearold bovine lenses, but he gives no indication of the changes occurring with growth. Salit2 found the water content of the rabbit lens to be 59.25 percent (the mean of only two cases), while Bruckner* reports
The eyes were removed from the animals within a few minutes of their death.* All
* From the Departments of Physiology and Ophthalmology, College of Medicine, State University of Iowa.
t The cattle eyes were obtained through the courtesy of Gay's Locker Plant, Iowa City, Iowa, and Wilson Packing Company, Cedar Rapids, Iowa.
the water content as 62.7 percent (also the mean of two cases). No report on the age of the animals was made by either worker. Field and others 5 found a mean dry weight of 30.7 percent, which would indicate a water content of 69.3 percent. Bruckner 4 reported the water content of a lens from a 5-day-old cat as 74.5 percent. No other references on the water content of cat lenses have been found in the literature. Therefore, it is important to make a more extensive study of the water content of the crystalline lens to correlate its relationship with the age of the animal in cattle, rabbits, and cats, three species commonly employed in experimental studies of the eye. METHODS
A. ROBBIE, P H . D . , AND P. J. LEINFELDER,
M.D.
Iowa City, Iowa
Cellular respiration processes are extremely important for both the functioning and survival of the cerebral cortex and the retina and, if the supply of either oxygen or substrate is interrupted, unconsciousness follows quickly. Manometric studies on the metabolism of isolated brain have revealed something of the nature of the enzyme systems that are involved in oxidation, and of the effects of anesthetics and other pharmacologie agents. A comparison of the metabolism of the cerebral cortex and the retina may be of interest to the ophthalmologist because of interest both in the retina itself and in the information that retinal studies may give about the physiology of the central nervous system. Physiologically, the retina resembles cerebral cortex in its high rate of oxygen consumption, its unusually high rate of both aerobic and anaerobic glycolysis,1 its dependence upon glucose substrate, and the almost complete depression of respiration produced by heavy metal inhibitors.2 Since the retina is derived from neuroectoderm, a morphologic similarity is to be expected, and this has been shown to be true up to the 45-mm. stage of embryologie development.3 In the adult tissues, in spite of specialization of each, a similarity remains in the lamellar arrangement of nerve cells, synaptic, and nerve-fiber layers. Isolated rat retina is a convenient tissue for use in manometric metabolism measurements. It is easily and quickly removed from the eye with relatively slight injury, and is thin enough to permit adequate oxygιnation without slicing, even though air is the gas phase rather than 100-percent oxygen. * From the Departments of Ophthalmology and Physiology, College of Medicine, State University of Iowa. Aided by a grant from the John and Mary R. Markle Foundation.
If the suspension medium is buffered and contains glucose and a properly balanced salt mixture, linear respiration may be obtained for a period of 5 to 7 hours. It is thus possible and convenient to measure respiration during a control, experimental, and recovery period on the same tissue. Since the rat retina does not break up while it is shaking in the manometer, it may be easily removed and weighed at the end of the experiment. Experimental determination of respiration in isolated brain is complicated by the procedure of cutting thin slices of the tissue in order to insure adequate oxygιnation. Since the nerve cells are extensively branched, it is evident that a slice of optimum thinness must have a high proportion of cells with cut processes. It is recognized that cutting a nerve process may cause degeneration of the cell body even though the cell is otherwise undisturbed. In an artificially prepared saline solution, there may be additional injury from diffusion into the cell of ions in a concentration different from the normal constitution. Certain experimental agents that do not penetrate the intact cell may likewise adversely and misleadingly affect the brain slice. The necessity for use of 100-percent oxygen also complicates certain determinations because of the oxygen-poisoning effect. TECHNIQUE
The experiments described in the present paper were performed on albino rats. The animals were killed by breaking the neck, and either the eyes or the brain was removed immediately. In studies on cerebral cortex, the brain was sliced by means of a razor blade and a transparent plastic template.4 The slices were weighed on a microtorsion balance before they were immersed in physi-
208
RETINA IN METABOLISM STUDIES ologic saline solution. Retinas were removed by cutting around the globe just posterior to the ora serrata. The retinas were then lifted out and cut into halves to prevent folding. The isolated tissues were suspended in phosphate-buffered saline solution at pH 7.35 containing 0.2-percent glucose.5 Small Warburg manometer flasks containing a total volume of about 7 cc. were used for the oxygen consumption determinations. The center wells contained 10-percent KOH on filter paper. Temperature of the water bath was 37.2°C. Unless otherwise mentioned, experiments on retinas were performed with air as the gas phase and those on brain slices with 100-percent oxygen. Retinal weights were determined after rinsing the tissues at the end of the experimental periods and drying them on weighed cover glasses at 110°C. The dry weights for the brain slices were calculated from the recorded wet weights by means of a wetdry ratio obtained by drying sample tissue at 110°C. . In the histologie studies, brain tissue was prepared in the following manner: For the control sections, Figure 5 (left), the brain was removed immediately after the death of the animal, and slices of the cerebral cortex were cut and placed in 10-percent neutral formalin for fixation. In other specimens studied, slices of tissue were placed in manometer flasks in the phosphate-glucose-saline solution. These flasks were then placed in the water bath and the respiration rate was recorded for one-half and 2j4-hour intervals in order to verify the viability of the tissue. Following this, the tissue was removed from the flask and fixed. A similar procedure was used in experiments on the rat retina. In addition, tissue slices and blocks from rat brain were stored in saline solution at 4°C. for intervals varying from 2 to 24 hours. At the end of the storage period, the brain tissue was placed in formalin for fixa-
209
tion. Sections made from all preparations were stained with hematoxylin and eosin. EXPERIMENTAL RESULTS AND DISCUSSION
Results of a number of experiments seem to indicate that in the tissues studied oxygen consumption is not dependent upon the integrity of the cell. Both cortex slices or chopped tissue made by cutting the cortex into small pieces respire at about the same level. Similarly, either intact retina or retina which has been cut into small pieces shows the same oxygen uptake for a period of at least one hour. However, if the tissues are ground up in a tissue grinder, then the respiration falls off considerably. It is possible that as long as the gross intracellular organization is not appreciably disturbed, the respiratory enzyme systems may function at a normal rate. Figure 1 shows that when a properly buffered and balanced saline solution containing glucose is used, the respiration of the retina in air is maintained at a constant rate for as long as 7 hours. The lower curve in the figure shows the course of oxygen consumption when a retina is run in fluid containing no glucose. The rapid decline in respiration to almost the zero level is convincing evidence that the process being measured in the respirometers is glucose metabolism rather than any sort of autolytic degenerative change. There is a pronounced difference in the course of oxygen consumption of the retina in air and in 100-percent oxygen. In air, the rate of respiration is constant during the observation period. In oxygen, the rate of respiration is higher at the start of the measurement period, but there is a marked falling off and, by the time the experiment has been run for a period of 7 hours, the oxygen consumption has decreased to 50 percent of that of the specimen in air. This is shown in Figure 1. The curve for brain respiration is similar to that for retina in oxygen and it may be
210
W. A. ROBBIE AND P. J. LEINFELDER
that if it were possible to run cortex slices in air, the respiration would be more constant. This is not feasible since a tissue slice that is thin enough to permit adequate oxygιnation at the center, with air as the gas phase, is almost impossible to prepare. The steady rate of respiration of the retina in air perhaps more nearly resembles the situation in the living animal, since the oxygen tension within the eye can certainly
0
tex slices, the response of the injured cells to certain chemical factors may be considerably different than would be expected from normal tissue. It is well known that highly dissociated acids do not go through the normal cell membrane. Figure 2 shows the results of an experiment with brain slices and retinas in which the tissues were exposed to a saline solution at pH 5.2 for a period of one hour, after
Z
A-
6
HOURS Fig. 1 (Robbie and Leinfelder). Oxygen consumption of isolated rat retinas in phosphate-buffered saline solution containing 0.2 percent glucose. Ordinate shows the mm.3 of 0 2 consumed per mg. dry weight, and the abscissa the time in hours.
never approach that which exists in the flasks gassed with .100-percent oxygen. Whether the increase in oxygen consumption of the retina in oxygen at the start of the measurement period is actual respiration or simply oxidation of substrates that might not otherwise be utilized, is a question. The falling· off in respiration at the end of the 6- or 7-hour period of measurement may possibly be an oxygen-poisoning effect. Although the rate of respiration is perhaps not immediately affected by cutting the processes of the nerve cells in cerebral cor-
which the recovery oxygen consumption was measured. Although the retina has apparently not been damaged by this exposure to an acid environment, the cortex slices with their· cut cells and processes show considerable injury, as evidenced by a marked decline in respiration. Kidney and liver slices also have been shown to recover almost completely after an hour's exposure to a medium at pH 5.6 Figure 3 shows the results of an experiment using 0.1 M potassium chloride in the suspension fluid. In vivo, the potassium ion
211
RETINA IN METABOLISM STUDIES penetrates the brain very slowly.7 Yet the respiration of the brain slices is increased by almost 100 percent when a high concentration of potassium is included in the saline solution. (The sodium chloride concentration in these experiments was reduced to maintain an isotonic medium.) The retina, on the other hand, shows, if anything, a slight decrease in oxygen consumption in the medium containing high potassium. Similar observations have been reported by Dickens.5 This again indicates that potassium, whatever may be the nature of its stimulating action, apparently does get into the cortex cells which have been cut, but not into the retina cells. The cytologie picture also gives evidence that the brain is much more injured during
With brain tissue, on the other hand (fig. 5), edema was noted after one-half hour in the Warburg flasks, and the ganglion cells had begun to undergo chromatolytic change, as shown by the swelling of the
16
o
«4-
A
/
«V / y
30
o ιο
0
*.·
*?/
éf
/
^Ã
*w
# #
1
/
y
M" Z
"
0 HOURS
1
I
40
O MINUTES
40
Fig. 3 (Robbie and Leinfelder). Effect of addition of 0.1M. KC1 to phosphate-buffered saline solution on the respiration of retina and cerebral cortex slice. Ordinate : mm." of Oδ per mg. dry weight.
BRAIN
RETINA
.-'
<
20
40·
BRAIN
«TINA
<<***..
1
2
Fig. 2 (Robbie and Leinfelder). Recovery of retinas and cerebral cortex slices after one hour at pH 5.2. During the exposure period the tissues were shaken in manometer flasks in glucose-saline solution containing 0.01M. primary sodium phosphate. Ordinate shows oxygen uptake during recovery period in mm." Oj per mg. dry weight. preparation for respiration measurement than the retina. Figure 4 shows sections from a control retina and one which had been run for 2% hours in a manometer flask at 37.2° C. The ganglion cells and the other retinal cellular elements were similar in both the control and in the experimental tissues at the end of the experiment.
cytoplasm and nuclei. In some instances, the ganglion cell nuclei were extremely vacuolated, indicating karyorrhexis. These changes were considerably exaggerated in a 2J^-hour specimen and many of the ganglion cells had disappeared, while all of those remaining showed advanced chromatolysis. In some instances, glial clumping about the ganglion cells could be observed. A similar type of change was observed after two hours' storage at 4°C, and this became increasingly apparent after longer storage periods. When the brain remains intact in the dead animal at room temperature for as long as ΐÕæ hours, chromatolytic changes do not occur. However, if the brain is removed and tissue slices or blocks are prepared, evidence of cellular damage can be recognized within half an hour if the tissue is maintained at 37° C. ; similar changes are apparent after two hours when the tissue is kept at 4°C. After greater periods of time, more severe
212
W. A. ROBBIE AND P. J. LEINFELDER
Fig. 4 (Robbie and Leinfelder). Photomicrographs of sections of rat retinas. (Left) Control section. (Right) Section of retina shaken for 2 ^ hours in manometer flask at 37.2° C.
Fig. 5 (Robbie and Leinfelder). Sections of rat cerebral cortex slices. Control on left fixed immediately after death. Experimental tissue on right shaken for one-half hour in manometer flask before fixation.
RETINA IN METABOLISM STUDIES
chromatolytic phenomena are apparent. These changes, which are identical with those occurring in encephalomalacia, appear to be the result of injury to the ganglion cells or their processes, and the chromatolytic response may occur directly as a result of the injury to the cells or indirectly because of the change in the intracellular chemical environment that occurs when the cut cells are placed in physiologic saline solution. (The concentrations of certain ions in protoplasm are quite different than those in blood plasma.) In the preparation of retinal tissue, the ganglion cells themselves are not directly injured and only the axone is cut. This injury to the retinal ganglion cell axone occurs at some distance from the cell body, and is less severe trauma than that which occurs to the ganglion cells of the brain when tissue slices are prepared, for, in the latter case, both cell processes and the cells themselves are cut. With the brain slices, a much greater proportion of the cell cytoplasm is exposed to an abnormal chemical environment than in the retina when the main mass of the tissue has not been disrupted. CONCLUSIONS
The structural and physiologic similarity between the cerebral cortex and the retina
213
make it seem probable that the respiratory response of the retina is, in many ways, truly indicative of central nervous system metabolism. Rat retinas are convenient to prepare and to use in manometric determinations, and the period of constant oxygen uptake is long enough to allow measurement of experimental treatment periods and recovery in the same tissue. Injury to the cell membrane in cerebral cortex slices is evidenced by hydrogen ion effect and potassium stimulation of respiration, as well as by a rapid cytologie response to the injury. In studies involving an agent that normally does not penetrate the brain cell, results may be more representative if measurements are made on the retina rather than on a brain slice. The toxic action of pure oxygen on the retina, and the similarity of the curves representing respiration in oxygen of the retina and the brain, also indicate that brain metabolism in 100-percent oxygen is probably not normal. It is believed that studies of the retina may in some ways be more clearly indicative of central nervous-system metabolism than observations on brain slices, and it may be advisable to use the retina for comparison and control experiments. University Hospitals.
REFERENCES
1. Warburg, O. : άber die Klassifizierung tierischer Gewebe nach ihrem Stoffwechsel. Biochem. Ztschr., 184:484,1927. 2. Robbie, W. A., and Leinfelder, P. J. : Cyanide inhibition of retinal respiration in bicarbonate buffer. Arch. Biochem., 16:437,1947. 3. Haden, H. C. : Concerning the similarity of the developing retina and brain wall in human embryos. Am. J. Ophth., 28:943, 1945. 4. Fuhrman, F. A., and Field, J., 2d: Action of diphenyloxazolidinedione on brain respiration at varied temperature levels. J. Pharmacol. & Exper. Therap., 77:229,1942. 5. Dickens, F., and Greville, G. D. : CLXXVII. The metabolism of normal and tumour tissue. XIII. Neutral salt effects. Biochem. J., 29:1468,1935. 6. Stearns, A. W., Jr., Greenblatt, M., Canzanelli, A., and Rapport, D.: The reversibility of pH effects on the ft consumption of tissues. Am. J. Physiol., 132:564,1941. 7. Fenn, W. O. : The role of potassium in physiological processes. Physiol. Rev., 20:377, 1940.
214
W. A. ROBBIE AND P. J. LEINFELDER DISCUSSION
DR. T. D. DUANE (Iowa City, Iowa) : Granit in Sweden, Adrian in England, Bronk, Hartline in the United States have shown that the retina is an ideal tissue upon which to study electrical phenomena in the body, action potential currents, secondary to stimulation, and so forth. Dr. Robbie has just pointed out that the retina is an ideal tissue upon which to study metabolic activities of the central nervous system. It seems to me this is a typical example of the point which Dr. Friedenwald was making last night, that we are opening new vistas in research in ophthalmology, because' the next obvious step is to make a correlation between electrical phenomena and metabolic phenomena. In other words, it seems conceivable to me that one might injure the various portions of the retina, say the rods and cones, bipolars, or the ganglion cells, to see what effect that has upon the electrical phenomena in the electroretinogram, and then the retina could be removed and studied in vitro from the metabolic standpoint and I think some very interesting correlation might be made. DR. P. J. LEINFELDER (Iowa City, Iowa) : The portion of this paper that has been particularly interesting and spectacular to me is the portion dealing with the pathology in the nervous system. Unfortunately, the lantern slides do not show the great degree of chromatolysis that occurs in the ganglion cells of the brain. You who have taken photomicrographs of brain tissue at 800 or 900 magnification know how difficult it is to show representative areas in a particular slide. However, the effects of slicing the brain tissue are real and occur with great rapidity for they can be clearly recognized one-half hour after sectioning. The paradox of the situation is this. If we leave the brain intact in the animal for 2,4,6 hours, and then remove the cal-
varium, take out the brain, take a thin slice of it, and put it in fixative immediately, we obtain perfectly normal histologie appearances. However, if the brain tissue is sliced and then allowed to lie in saline buffer solution with glucose and oxygen available, the changes of chromatolysis take place. Such changes are not recognized in the retina up to 6 hours when the temperature is 37.5°C, nor up to 24 hours when the retinas are kept at 4°C. The occurrence of these chromatolytic changes in nerve tissue which is assumed to be dead is a particularly interesting problem and one which has to be investigated in the light of what is taking place in the tissues as far as respiration, glycolysis, and utilization of substrate is concerned. DR. DAVID G. COGAN (Boston, Massachu-
setts) : I would like to ask Dr. Robbie if there is any contradiction in the apparent susceptibility of the retina to anoxemia, as judged by "black-outs," and the apparent resistance of the retina to oxygen lack as determined by studies in the Warburg apparatus. DR. ROBBIE (closing) : I would like to say that I have no way of knowing what effect the proteolytic enzymes may have on the histologie structure of the tissue. To Dr. Cogan's comment I may say that I think the two factors are possibly quite dissociable —oxygen consumption and the preservation of the visual functions. It is well known of course that the anoxic retina loses its power of perceiving light very rapidly, and anoxia for a period of 7 minutes in the intact eye leads to permanent blindness. Yet if we kill the animals and keep the dead bodies at 37° C. for a period of 45 minutes and then take the retinas out and measure them in the manometer flasks, the oxygen consumption is apparently normal. So it seems as if the limiting element in the system is something other than a chain of respiratory enzymes.
METABOLISM OF THE CRYSTALLINE LENS It may be that the ganglion cells themselves are particularly susceptible in some way morphologically or chemically. To Dr. Duane's contribution, I would like to discourse somewhat on his use of the word "ideal." We do not believe that the retina is necessarily ideal for these purposes. For example, Dr. Friedenwald suggested to me that perhaps the pigment layer is important in retinal function. When we take the retina out of the eye and leave the pigment layer behind, we no longer have a completely normal retina, and perhaps we are disturbing the function in that way. Furthermore, when an animal is killed, unless the eye is fixed immediately, the rod and cone
215
layer quickly becomes diffused in appearance. So the retinas we are studying probably do not have an intact rod and cone layer. What we are measuring is the metabolism of the surviving cells. Any conclusions regarding vision that involve the photochemistry of visual purple are perhaps not shown by these studies. The one point that I do wish to make is that possibly the retina is more representative of the normal central nervous system than the cerebral cortex slice, and at least it may be valuable to compare both types of tissues when the effect of a drug is being studied so that it may be possible to evaluate the factors of permeability and injury.
METABOLISM O F T H E CRYSTALLINE L E N S * I. WATER CONTENT AND GROWTH RATE LAWRENCE O. ELY,
M.D.
Iowa City, Iowa Water is quantitatively the major constituent found in the crystalline lens. Accurate interpretation of certain lenticular properties, namely, Q 0 2 , is dependent upon a precise knowledge of the ratio of wet weight to dry weight. Bellows1 presents a table summarizing the information in the literature concerning the water content of bovine lenses (Table 1 ), but it is of note that the cases cited by any one worker are too few in number to be of statistical significance except for Salit's2 figure of 65.41-percent water for 1- to 4-year-old bovine lenses. However, Krause 3 gives a statistically reliable water content of 67 percent in 1-yearold bovine lenses, but he gives no indication of the changes occurring with growth. Salit2 found the water content of the rabbit lens to be 59.25 percent (the mean of only two cases), while Bruckner* reports
The eyes were removed from the animals within a few minutes of their death.* All
* From the Departments of Physiology and Ophthalmology, College of Medicine, State University of Iowa.
t The cattle eyes were obtained through the courtesy of Gay's Locker Plant, Iowa City, Iowa, and Wilson Packing Company, Cedar Rapids, Iowa.
the water content as 62.7 percent (also the mean of two cases). No report on the age of the animals was made by either worker. Field and others 5 found a mean dry weight of 30.7 percent, which would indicate a water content of 69.3 percent. Bruckner 4 reported the water content of a lens from a 5-day-old cat as 74.5 percent. No other references on the water content of cat lenses have been found in the literature. Therefore, it is important to make a more extensive study of the water content of the crystalline lens to correlate its relationship with the age of the animal in cattle, rabbits, and cats, three species commonly employed in experimental studies of the eye. METHODS