Cell and Tissue Research
Cell Tissue Res. 198, 137-144 (1979)
9 by Springer-Verlag 1979
Effects of Halothane on Microtubules in the Sciatic Nerve of the Rat A. Livingston and G.A.Vergara Department of Pharmacology, Medical School, University Walk, Bristol, U.K.
Summary. The effect of halothane and colchicine on microtubule repolymerisation after exposure to cold has been examined in the sciatic nerve of the rat. Myelinated axons showed a significant decrease in microtubule numbers and density after exposure to both 2 0 m M halothane and 1 0 - S M colchicine; exposure to both agents simultaneously produced a further significant reduction when compared to halothane alone. It is suggested that halothane can interfere with microtubule repolymerisation in mammalian axons possibly in a similar way to that described in invertebrates. Key words: Microtubules - H a l o t h a n e - Sciatic nerve (Rat) -Repolymerisation. Many compounds have been shown to exert effects on the microtubules of peripheral nerves in mammals, and several groups of workers have examined the effects of halothane (2-bromo, 2-chloro, 1:1:1 trifluoroethane), a volatile general anaesthetic, on these organelles. These earlier investigations were prompted by the suggestion of Allison and N u n n (1968) that general anaesthetics might exert their effects by causing depolymerisation of microtubules in neurones and hence by interference with their neuronal properties. The studies of Hinkley and Green (1971) on rabbit vagus nerve suggested that halothane had effects on the conductivity of the nerve and that there was some increase in the numbers of microtubules present; Kennedy et al. (1972) studied the effects of halothane on rapid axonal transport in rabbit vagus and could find no effects, and examination of microtubule numbers indicated very little effect except at high concentration when a slight reduction in numbers was noted. Saubermann and Gallagher (1973) examined the effects of inhaled halothane on microtubule numbers in mouse optic nerve and found no effects. However, the studies of Hinkley and Samson (1972) and Send offprint requests to: Dr. A. Livingston,Department of Pharmacology,Medical School, University
Walk, Bristol BS8 1TD, U.K. We are very grateful to K. Barnes for skilled technical assistance and to Dr. S. Lawson for advice on the measurement of axonal areas. Acknowledgements:
0302-766X/79/0198/0137/$01.60
138
A. Livingston and G.A.Vergara
H i n k l e y (1976) d e m o n s t r a t e d clearly that h a l o t h a n e exerted effects o n the m i c r o t u b u l e s o f crayfish connectives to induce m a c r o t u b u l e f o r m a t i o n , a n effect which h a d been d e m o n s t r a t e d in various m i c r o t u b u l e systems with other agents such as colchicine (Tilney, 1968), vinblastine (Tyson a n d Bulgar, 1973) a n d i s o p r o p y l - N - p h e n y l c a r b a m a t e (Hanzely a n d Olah, 1970). I n view of the effects d e m o n s t r a t e d on invertebrate n e u r o n a l m i c r o t u b u l e s it was decided to re-examine the effects of h a l o t h a n e o n m a m m a l i a n n e u r o n a l m i c r o t u b u l e s particularly with respect to its effects o n repolymerisation.
Materials and Methods Adult male Wistar rats were killed by decapitation and the sciatic nerves dissected out, desheathed and cut into 3 mm lengths. The portions of nerves were divided into four groups and placed in gas tight bottles containing 5 ml of Eagles 199 medium which had been gassed with 95 % O2:5 % CO2 previously. The bottles were then incubated for 30min at 0~ to produce depolymerisation of the microtubules, followingthis they were then incubated for 30 min at 37~C, one group were incubated with no additions to the medium, one group with the addition of halothane (20 raM), one group with the addition of colchieine (I 0-5 M) and the other group with both halothane (20 mM) and colchicine (10-5 M). Following incubation the segments of nerve were removed from the medium and fixed for 2 h in 2.5 % glutaraldehyde pH 7,2 in caeodylate buffer. They were rinsed in buffer and post-fixed in 1% osmium tetroxide, pH 7.2 in cacodylate buffer, for 11/2h. The segmentswere then rinsed in water and dehydrated in graded ethanols and epoxypropane and embededin Araldite. Transversesectionswere cut on a Porter-Blum MT2B ultramicrotome and examined at 75kV in a Hitachi HU12A electron microscope. Random electron micrographs of myelinated axons were taken at 10,000x magnification and the number of mierotubules per axon counted from enlarged prints. The counting procedures were done by a double blind technique and at least thirty prints from each group were examined. In addition the areas of the axons were measured using a Graf pen interfaced with a Tektronix 4051 minieomputor and the numbers of microtubules per unit axonal area were calculated.
Results The i n c u b a t i o n procedure used tended to cause some tissue damage which was usually reflected in d i s t o r t i o n of the myelin sheaths of the nerves examined. There did n o t a p p e a r to be a n y difference in the degree o f d a m a g e seen in the four groups examined, which suggested that the i n c u b a t i o n procedure rather t h a n the presence o f h a l o t h a n e or colchicine caused the distortion o f myelin. The control (untreated) axons showed a good preservation of axonal contents with m i c r o t u b u l e s being clearly visible (Fig. 1), the axons from the g r o u p treated with 1 0 - 5 M colchicine showed a considerable reduction in the n u m b e r of intact microtubules, b u t little a l t e r a t i o n in the n a t u r e o f the filamentous n e t w o r k (Fig. 2). The axons from the nerves treated with 2 0 r a M h a l o t h a n e also showed a decrease in the n u m b e r o f intact microtubules, b u t again the filamentous n e t w o r k showed a n o r m a l a p p e a r a n c e (Fig. 3). The axons treated with b o t h h a l o t h a n e a n d colchicine showed a r e d u c t i o n in m i c r o t u b u l e s with a n a p p a r e n t l y n o r m a l filamentous c o m p l e m e n t (Fig. 4). The c o u n t i n g o f m i c r o t u b u l e s per axon supported the impressions gained by e x a m i n i n g the micrographs. Figure 5 shows the results expressed as the m e a n n u m b e r o f m i c r o t u b u l e s per a x o n from the four groups. It can be seen that all three
Fig. 1. Section ofmyelinated axon from rat sciatic nerve incubated for 30 min at 0 ~ C and 30 min at 37 ~ C, showing n u m e r o u s microtubules (large arrows) and neurofilaments (small arrows), x 16,000 Fig. 2. Section of myelinated axon from rat sciatic nerve treated with 1O- ~ M colchicine during the repolymerisation incubation, showing large numbers o f neurofilaments (small arrows) and very few microtubules (large arrows). • 16,000
Fig. 3. Section of myelinated axon from rat sciatic nerve treated with 20 mM halothane during the repolymerisation incubation, showing large numbers of neurofilaments (small arrows) and occasional microtubules (large arrows), x 16,000 Fig. 4. Section of myelinated axon from rat sciatic nerve treated with 20 mM halothane and 10-5M colchicine during the repolymerisation incubation, showing large numbers of neurofilaments (small arrows) and occasional microtubules (large arrows), x 16,000
Halothane and Microtubules
141
q Significonce of difference between groups
~
"I
Significance of difference between groups
Cont. HaL. Cot, Hal.+[o[.
30=25-
o o20-
1,/+-
Cont 12Cot,
=
Hol.~-Cot
~~ O,B-
0.6-
215-
~
Cont. Hot. CoL. Hot.+Cot Cont Hal. CoL, Hab-Cot
~= 0,2Control
Halofhane Col.chicine Holofhone 20raM 20ram
1ffsM
+ Co~chicine 1(]sM
0
Control Hatofhone Eotchicine Hatothane 20raM 2Dram
I0-5M
+Co[chicine10-SM
Fig. 5. Diagram to show the mean number ofmicrotubules per axon (+ S.E.M.) from the four groups of axons examined. The P values for significance of difference between the groups are shown Fig. 6. Diagram to show the mean number of microtubules per square l~m of axon (S.E.M.) for the four groups of axons. The P values for the significance of difference between the groups are shown
treatments, halothane, colchicine and halothane+ colchicine, resulted in a significant decrease in microtubules, and that the extent of the decrease was larger for colchicine than halothane, and larger still with the combination of treatments. In fact, the addition ofcolchicine significantly lowered the effects of halothane. The examination of the results in terms of the number of microtubules per unit axonal area displayed broadly the same picture (Fig. 6), with again all three treated groups showing significantly lower values than the controls, and the halothane + colchicine treatment leading to significantly lower values than the halothane treatment. Higher power examination of the axonal contents confirmed the impressions gained from Figs. 1 4 ; the control sections showed apparently normal microtubules (Fig. 7) whilst the halothane, colchicine and halothane + colchicine treated samples showed numerous 10 nm filaments but few microtubules (Figs. 8-10).
Discussion The incubation of portions of rat sciatic nerve with halothane resulted in a reduction in the number of microtubules seen in transverse sections of myelinated axons. The effect of 20 mM halothane was roughly similar to that produced by 10-s M colchicine under the experimental conditions used. The variation in the responses of neuronal microtubules to halothane previously reported (Hinkley and Green, 1971 ; Kennedy et al., 1972; Saubermann and Gallagher, 1973), compared to the responses reported by Hinkley (1976) in crayfish axons, suggested that a closer examination of these effects was indicated. In his experiments Hinktey (1976) predicted that halothane would produce an initial
142
A. Livingston and G.A. Vergara
Fig. 7. Section from axon of control sciatic nerve showing numerous microtubules and neurofilaments. x 116,000 Fig.8. Section from axon o f colchicine-treated sciatic nerve showing neurof'llaments, x 116,000
Halothane and Microtubules
143
Fig.9. Section from axon o f halothane-treated sciatic nerve showing neurofilaments, x 116,000 Fig. 10. Section from axon o f halothane- and colchicine-treated nerve showing neurofilaments. x 116,000
144
A. Livingston and G_A_.Vergara
dissociation o f microtubules before the macrotubule production. Since the depolymerisation and repolymerisation o f microtubules is a dynamic situation, it was decided to examine the effects o f halothane on just one step o f the process, namely the repolymerisation that occurs during incubation at 37~ following depolymerisation caused by exposure to cold. U n d e r these conditions halothane caused a significant decrease in the numbers o f microtubules, presumably by affecting the repolymerisation process. The effect was quantitatively similar to that p r o d u c e d by colchicine, but addition o f colchicine to halothane significantly reduced the n u m b e r s still further, which might indicate a different mechanism o f action. F u r t h e r experiments will be required to examine this point. It was n o t possible to demonstrate the formation o f m a c r o t u b u l e s by halothane in this series o f experiments; however, the m e t h o d o l o g y o f Hinkley was not followed exactly in these experiments and it m a y be that prewarming for 1 h at 40 ~ C m a y be necessary to produce these effects. The d e m o n s t r a t i o n o f the prevention o f microtubule repolymerisation by halothane in this study supports the observations o f other workers on the effects o f halothane on microtubules f r o m n o n - m a m m a l i a n tissues.
References Allison, A.C., Nunn, J.F.: Effects of general anaesthetics on microtubules. Lancet, 2, 1326-1329 (1968) Hanzely, L., Olah, L.V.: Digitonin-induced formation of a new tubular element in dividing root tip cells of Allium sativum. J. Cell Biol. 47, 82a (1970) Hinkley, R.E.: Microtubule-macrotubule transformations induced by volatile anesthetics. J. Ultrastr. Res. 57, 237-250 (1976) Hinkley, R.E., Green, L.S.: Effects ofhalothane and colchicine on microtubules and electrical activity of rabbit vagus nerve. J. Neurobiol. 2, 97-105 (1971) Hinkley, R.E., Samson, F.E.: Anesthetic induced transformation of axonal microtubules. J. Cell. Biol. 53, 258-263 (1972) Kennedy,R.D., Fink, B.R., Byers, M.R.: The effect of halothane on rapid axonal transport in the rabbit vagus. Anesthesiology 36, 433-443 (1972) Saubermann, A.J., Gallagher, M.L.: Mechanisms of general anesthesia: Failure of pentobarbital and halothane to depolymerise microtubules in mouse optic nerve. Anesthesiology 38, 25-29 (1973) Tilney, L.G.: The effect of colchicine on the formation and maintenance of the axopodia and the redevelopment of pattern in Actionsphaerium nucleofilum J. Cell Sci., 3, 549-562 (1968) Tyson, G.E., Bulgar, R.E.: Vinblastine induced paracrystals and unusually large microtubules (macrotubules) in rat renal cells. Z. Zellforsch. 141, 443-458 (1973) Accepted February 5, 1979
Cell Tissue Res. 198, 137-144 (1979)
9 by Springer-Verlag 1979
Effects of Halothane on Microtubules in the Sciatic Nerve of the Rat A. Livingston and G.A.Vergara Department of Pharmacology, Medical School, University Walk, Bristol, U.K.
Summary. The effect of halothane and colchicine on microtubule repolymerisation after exposure to cold has been examined in the sciatic nerve of the rat. Myelinated axons showed a significant decrease in microtubule numbers and density after exposure to both 2 0 m M halothane and 1 0 - S M colchicine; exposure to both agents simultaneously produced a further significant reduction when compared to halothane alone. It is suggested that halothane can interfere with microtubule repolymerisation in mammalian axons possibly in a similar way to that described in invertebrates. Key words: Microtubules - H a l o t h a n e - Sciatic nerve (Rat) -Repolymerisation. Many compounds have been shown to exert effects on the microtubules of peripheral nerves in mammals, and several groups of workers have examined the effects of halothane (2-bromo, 2-chloro, 1:1:1 trifluoroethane), a volatile general anaesthetic, on these organelles. These earlier investigations were prompted by the suggestion of Allison and N u n n (1968) that general anaesthetics might exert their effects by causing depolymerisation of microtubules in neurones and hence by interference with their neuronal properties. The studies of Hinkley and Green (1971) on rabbit vagus nerve suggested that halothane had effects on the conductivity of the nerve and that there was some increase in the numbers of microtubules present; Kennedy et al. (1972) studied the effects of halothane on rapid axonal transport in rabbit vagus and could find no effects, and examination of microtubule numbers indicated very little effect except at high concentration when a slight reduction in numbers was noted. Saubermann and Gallagher (1973) examined the effects of inhaled halothane on microtubule numbers in mouse optic nerve and found no effects. However, the studies of Hinkley and Samson (1972) and Send offprint requests to: Dr. A. Livingston,Department of Pharmacology,Medical School, University
Walk, Bristol BS8 1TD, U.K. We are very grateful to K. Barnes for skilled technical assistance and to Dr. S. Lawson for advice on the measurement of axonal areas. Acknowledgements:
0302-766X/79/0198/0137/$01.60
138
A. Livingston and G.A.Vergara
H i n k l e y (1976) d e m o n s t r a t e d clearly that h a l o t h a n e exerted effects o n the m i c r o t u b u l e s o f crayfish connectives to induce m a c r o t u b u l e f o r m a t i o n , a n effect which h a d been d e m o n s t r a t e d in various m i c r o t u b u l e systems with other agents such as colchicine (Tilney, 1968), vinblastine (Tyson a n d Bulgar, 1973) a n d i s o p r o p y l - N - p h e n y l c a r b a m a t e (Hanzely a n d Olah, 1970). I n view of the effects d e m o n s t r a t e d on invertebrate n e u r o n a l m i c r o t u b u l e s it was decided to re-examine the effects of h a l o t h a n e o n m a m m a l i a n n e u r o n a l m i c r o t u b u l e s particularly with respect to its effects o n repolymerisation.
Materials and Methods Adult male Wistar rats were killed by decapitation and the sciatic nerves dissected out, desheathed and cut into 3 mm lengths. The portions of nerves were divided into four groups and placed in gas tight bottles containing 5 ml of Eagles 199 medium which had been gassed with 95 % O2:5 % CO2 previously. The bottles were then incubated for 30min at 0~ to produce depolymerisation of the microtubules, followingthis they were then incubated for 30 min at 37~C, one group were incubated with no additions to the medium, one group with the addition of halothane (20 raM), one group with the addition of colchieine (I 0-5 M) and the other group with both halothane (20 mM) and colchicine (10-5 M). Following incubation the segments of nerve were removed from the medium and fixed for 2 h in 2.5 % glutaraldehyde pH 7,2 in caeodylate buffer. They were rinsed in buffer and post-fixed in 1% osmium tetroxide, pH 7.2 in cacodylate buffer, for 11/2h. The segmentswere then rinsed in water and dehydrated in graded ethanols and epoxypropane and embededin Araldite. Transversesectionswere cut on a Porter-Blum MT2B ultramicrotome and examined at 75kV in a Hitachi HU12A electron microscope. Random electron micrographs of myelinated axons were taken at 10,000x magnification and the number of mierotubules per axon counted from enlarged prints. The counting procedures were done by a double blind technique and at least thirty prints from each group were examined. In addition the areas of the axons were measured using a Graf pen interfaced with a Tektronix 4051 minieomputor and the numbers of microtubules per unit axonal area were calculated.
Results The i n c u b a t i o n procedure used tended to cause some tissue damage which was usually reflected in d i s t o r t i o n of the myelin sheaths of the nerves examined. There did n o t a p p e a r to be a n y difference in the degree o f d a m a g e seen in the four groups examined, which suggested that the i n c u b a t i o n procedure rather t h a n the presence o f h a l o t h a n e or colchicine caused the distortion o f myelin. The control (untreated) axons showed a good preservation of axonal contents with m i c r o t u b u l e s being clearly visible (Fig. 1), the axons from the g r o u p treated with 1 0 - 5 M colchicine showed a considerable reduction in the n u m b e r of intact microtubules, b u t little a l t e r a t i o n in the n a t u r e o f the filamentous n e t w o r k (Fig. 2). The axons from the nerves treated with 2 0 r a M h a l o t h a n e also showed a decrease in the n u m b e r o f intact microtubules, b u t again the filamentous n e t w o r k showed a n o r m a l a p p e a r a n c e (Fig. 3). The axons treated with b o t h h a l o t h a n e a n d colchicine showed a r e d u c t i o n in m i c r o t u b u l e s with a n a p p a r e n t l y n o r m a l filamentous c o m p l e m e n t (Fig. 4). The c o u n t i n g o f m i c r o t u b u l e s per axon supported the impressions gained by e x a m i n i n g the micrographs. Figure 5 shows the results expressed as the m e a n n u m b e r o f m i c r o t u b u l e s per a x o n from the four groups. It can be seen that all three
Fig. 1. Section ofmyelinated axon from rat sciatic nerve incubated for 30 min at 0 ~ C and 30 min at 37 ~ C, showing n u m e r o u s microtubules (large arrows) and neurofilaments (small arrows), x 16,000 Fig. 2. Section of myelinated axon from rat sciatic nerve treated with 1O- ~ M colchicine during the repolymerisation incubation, showing large numbers o f neurofilaments (small arrows) and very few microtubules (large arrows). • 16,000
Fig. 3. Section of myelinated axon from rat sciatic nerve treated with 20 mM halothane during the repolymerisation incubation, showing large numbers of neurofilaments (small arrows) and occasional microtubules (large arrows), x 16,000 Fig. 4. Section of myelinated axon from rat sciatic nerve treated with 20 mM halothane and 10-5M colchicine during the repolymerisation incubation, showing large numbers of neurofilaments (small arrows) and occasional microtubules (large arrows), x 16,000
Halothane and Microtubules
141
q Significonce of difference between groups
~
"I
Significance of difference between groups
Cont. HaL. Cot, Hal.+[o[.
30=25-
o o20-
1,/+-
Cont 12Cot,
=
Hol.~-Cot
~~ O,B-
0.6-
215-
~
Cont. Hot. CoL. Hot.+Cot Cont Hal. CoL, Hab-Cot
~= 0,2Control
Halofhane Col.chicine Holofhone 20raM 20ram
1ffsM
+ Co~chicine 1(]sM
0
Control Hatofhone Eotchicine Hatothane 20raM 2Dram
I0-5M
+Co[chicine10-SM
Fig. 5. Diagram to show the mean number ofmicrotubules per axon (+ S.E.M.) from the four groups of axons examined. The P values for significance of difference between the groups are shown Fig. 6. Diagram to show the mean number of microtubules per square l~m of axon (S.E.M.) for the four groups of axons. The P values for the significance of difference between the groups are shown
treatments, halothane, colchicine and halothane+ colchicine, resulted in a significant decrease in microtubules, and that the extent of the decrease was larger for colchicine than halothane, and larger still with the combination of treatments. In fact, the addition ofcolchicine significantly lowered the effects of halothane. The examination of the results in terms of the number of microtubules per unit axonal area displayed broadly the same picture (Fig. 6), with again all three treated groups showing significantly lower values than the controls, and the halothane + colchicine treatment leading to significantly lower values than the halothane treatment. Higher power examination of the axonal contents confirmed the impressions gained from Figs. 1 4 ; the control sections showed apparently normal microtubules (Fig. 7) whilst the halothane, colchicine and halothane + colchicine treated samples showed numerous 10 nm filaments but few microtubules (Figs. 8-10).
Discussion The incubation of portions of rat sciatic nerve with halothane resulted in a reduction in the number of microtubules seen in transverse sections of myelinated axons. The effect of 20 mM halothane was roughly similar to that produced by 10-s M colchicine under the experimental conditions used. The variation in the responses of neuronal microtubules to halothane previously reported (Hinkley and Green, 1971 ; Kennedy et al., 1972; Saubermann and Gallagher, 1973), compared to the responses reported by Hinkley (1976) in crayfish axons, suggested that a closer examination of these effects was indicated. In his experiments Hinktey (1976) predicted that halothane would produce an initial
142
A. Livingston and G.A. Vergara
Fig. 7. Section from axon of control sciatic nerve showing numerous microtubules and neurofilaments. x 116,000 Fig.8. Section from axon o f colchicine-treated sciatic nerve showing neurof'llaments, x 116,000
Halothane and Microtubules
143
Fig.9. Section from axon o f halothane-treated sciatic nerve showing neurofilaments, x 116,000 Fig. 10. Section from axon o f halothane- and colchicine-treated nerve showing neurofilaments. x 116,000
144
A. Livingston and G_A_.Vergara
dissociation o f microtubules before the macrotubule production. Since the depolymerisation and repolymerisation o f microtubules is a dynamic situation, it was decided to examine the effects o f halothane on just one step o f the process, namely the repolymerisation that occurs during incubation at 37~ following depolymerisation caused by exposure to cold. U n d e r these conditions halothane caused a significant decrease in the numbers o f microtubules, presumably by affecting the repolymerisation process. The effect was quantitatively similar to that p r o d u c e d by colchicine, but addition o f colchicine to halothane significantly reduced the n u m b e r s still further, which might indicate a different mechanism o f action. F u r t h e r experiments will be required to examine this point. It was n o t possible to demonstrate the formation o f m a c r o t u b u l e s by halothane in this series o f experiments; however, the m e t h o d o l o g y o f Hinkley was not followed exactly in these experiments and it m a y be that prewarming for 1 h at 40 ~ C m a y be necessary to produce these effects. The d e m o n s t r a t i o n o f the prevention o f microtubule repolymerisation by halothane in this study supports the observations o f other workers on the effects o f halothane on microtubules f r o m n o n - m a m m a l i a n tissues.
References Allison, A.C., Nunn, J.F.: Effects of general anaesthetics on microtubules. Lancet, 2, 1326-1329 (1968) Hanzely, L., Olah, L.V.: Digitonin-induced formation of a new tubular element in dividing root tip cells of Allium sativum. J. Cell Biol. 47, 82a (1970) Hinkley, R.E.: Microtubule-macrotubule transformations induced by volatile anesthetics. J. Ultrastr. Res. 57, 237-250 (1976) Hinkley, R.E., Green, L.S.: Effects ofhalothane and colchicine on microtubules and electrical activity of rabbit vagus nerve. J. Neurobiol. 2, 97-105 (1971) Hinkley, R.E., Samson, F.E.: Anesthetic induced transformation of axonal microtubules. J. Cell. Biol. 53, 258-263 (1972) Kennedy,R.D., Fink, B.R., Byers, M.R.: The effect of halothane on rapid axonal transport in the rabbit vagus. Anesthesiology 36, 433-443 (1972) Saubermann, A.J., Gallagher, M.L.: Mechanisms of general anesthesia: Failure of pentobarbital and halothane to depolymerise microtubules in mouse optic nerve. Anesthesiology 38, 25-29 (1973) Tilney, L.G.: The effect of colchicine on the formation and maintenance of the axopodia and the redevelopment of pattern in Actionsphaerium nucleofilum J. Cell Sci., 3, 549-562 (1968) Tyson, G.E., Bulgar, R.E.: Vinblastine induced paracrystals and unusually large microtubules (macrotubules) in rat renal cells. Z. Zellforsch. 141, 443-458 (1973) Accepted February 5, 1979
