EXPERIMENTAL
Reaction
NEUROLOGY
65, 87-98 (1979)
of the Mutant Mouse Nude to Axonal the Central Nervous System
GAD M. GILAD, VARDA H. GILAD,’ Laboratory
of Clinical
Science, Bethesda.
Received
National Maryland
December
of
J. KOPIN
AND IRWIN Institute 20014
Injuries
of Mental
Health,
21, 1978
The effects of unilateral forebrain and spinal cord hemisections were examined in the mutant athymic mouse nude. The activities of tyrosine hydroxylase and glutamic acid decarboxylase within the striatum and olfactory tubercle and within the midbrain ventral tegmentum, terminal fields of dopaminergic and GABA-ergic neurons, respectively, were measured to characterize the anterograde reaction of these neurons to axonal injuries. After forebrain hemisections a rapid (within 2 days) and permanent decrease was observed for all enzymes, indicating a permanent degeneration of axons with no recovery. Unilateral spinal cord hemisections resulted in a flaccid paralysis of the ipsilateral hind limb which subsided within 2 to 3 days after the lesion, and thereafter a permanent spasticity ensued. When spinal cord hemisection was repeated the initial flaccid paralysis did not occur and spasticity of the ipsilateral hind limb was immediately evident. The results indicate that regeneration of cut axons or even an improved motor recovery does not occur in the central nervous system of the nude mouse.
INTRODUCTION Regeneration of cut axons in the mammalian central nervous system is extremely limited and although some regenerative growth of cut axons occurs there, restitution of the original connections or functions has never been demonstrated. Several theories have been advanced (11, 15, 16) to explain this incapacity. One such theory, summarized recently (2), implicates immunological mechanisms: after injury, brain antigens reach the systemic circulation and result in antibody formation against brain Abbreviations: DA-dopaminergic, GABA--y-aminobutyric noradrenergic, TH-tyrosine hydroxylase, T-thymus-derived, decarboxylase. 1 Guest worker, Laboratory of Clinical Science.
acid-ergic, GAD-glutamic
NEacid
87 00 14-4886/79/070087-12$02.00/O Copyright 0 1979 by Academic Press, Inc. All rights of reproduction in any form reserved.
88
GILAD,
GILAD,
AND
KOPIN
proteins. To test this theory, we examined the reaction to axonal injury in the mutant nude mouse, which has an autosomal recessive trait resulting in a complete lack of cell-mediated and reduced humoral immunity (3, 15) and thus reduced immunoresistance (21, 25). Neurotransmitter synthesizing enzymes were used as specific markers to identify defined central neuronal systems (6,7, 17,22). After axonal injury of brain tracts known to contain catecholaminergic and GABA-ergic (GABA) fibers, changes in tyrosine hydroxylase (TH) and glutamic acid decarboxylase (GAD), specific markers for dopaminergic (DA) (5,7,8, 19) and GABA (6, 19,22) neurons, respectively, were examined in regions of brain in which these neurons terminate. In addition we studied the alterations in motor functions after spinal cord lesions. MATERIALS
AND METHODS
Animals. Experiments were conducted on outbred male and female homozygous nude (nu/nu) and heterozygous (Nu/+) mice (Sprague-Dawley, Madison, Wisconsin). Mice were housed five to a cage under germ-free conditions, fed ad libitum with sterilized standard laboratory chow and water, and maintained at 28°C with cycled lighting (on at 0900 h, off at 1700 h). Under these conditions animals appeared to be healthy and maintained normal body weight for the duration of the experiments. Characterization of Blood Lymphocytes. Lymphocytes were isolated on a Ficoll-Hypaque gradient (Winthrop Laboratories) and thymus-derived (T) lymphocytes were labeled by rosette formation with sheep erythrocytes according to the method of Bentwich et al. (1). The erythrocytes were pretreated enzymatically with neuraminidase (Bohring Diagnostics). Using this method we ascertained in a representative sample of animals the complete absence of T lymphocytes in nude mice and 30 to 40% T lymphocytes in heterozygous mice (Fig. 1). Cells were counted with a hemocytometer. Lesions. Unilateral hemisections were made in animals anesthetized with halothane (1.5% in 0,) under germ-free conditions. Brain or spinal cord was exposed by removing the bone with a dental drill. In brain, lesions were placed stereotaxically in the forebrain (1.8 mm posterior to bregma) to transect ascending DA fibers to the striatum and olfactory tubercle and noradrenergic (NE) fibers ascending to the cortex (23). In addition, such lesions transect descending GABA-producing fibers to the substantia nigra (6, 22). Brain lesions were made with a thin (co.5 mm), wide knife, made from a single-edge razor blade. In the spinal cord, lesions at the T5 level were made using a sharp segment of a double-edge razor blade mounted on a wood applicator stick.
CENTRAL
NERVOUS
SYSTEM
INJURY
IN THE
NUDE
MOUSE
89
90
GILAD,
GILAD, AND KOPIN
Groups of five or six operated animals and unoperated controls matched for age, gender, and strain were killed at various times after placement of the lesions, and the tissues were removed for biochemical analysis as described below. Assessment of Posture and Locomotion. After spinal hemisections animals were observed daily for changes in posture and locomotion. After surgery (groups of 12 animals) motion pictures of the animals were taken within 1 day and at 70 days after the lesion. Spasticity of the ipsilateral hind limb was most evident when the animal was trying to climb while being held by its tail. Changes in posture and locomotion produced by a second identical spinal cord lesion (in the original site) were examined in animals 70 days after the first lesion. Enzyme Assays. The animals were decapitated and their brains were rapidly removed, and the midbrain ventral tegmentum, olefactory tubercle, striatum, and frontal cortex were removed as previously described (7). The tissues were immediately frozen on dry ice and stored at -80°C. For assays, tissues were homogenized with a glass homogenizer in 10 vol (wt/vol) ice-cold 5 mM potassium phosphate buffer (PH 7.2) containing 0.1% Triton X-100 (voYvo1). Tyrosine dehydroxylase activity was assayed as described previously (19). Glutamic acid decarboxylase activity was assayed by the method of Roberts and Simonsen (20). RESULTS Forebrain Hemisections Regional Activities of Tyrosine Dehydroxylase (TH) and Glutamic Acid Decarboxylase (GAD) in the Brain. The regional distribution of TH activity
(Table 1) was similar to that previously reported in the rat (7), being about twice as high in the striatum and olefactory tubercle (which contain DA terminals) as in the midbrain ventral tegmentum (which contains DA cell bodies). Tyrosine dehydroxylase activity in the frontal cortex was low, consistent with the relatively low density of DA (14) and NE (23) terminals found in this region. There were no strain differences (Table 1). In this study postoperative enzyme changes in enzyme activity were compared in each animal using as controls the corresponding brain region on the contralateral side without lesion, in addition to homologous samples taken from unoperated control animals. In this manner it was ascertained that the unoperated side remained a stable point of reference. In unoperated animals there were no differences in enzyme activities between right and left sides of the brains (Table 1).
CENTRAL
NERVOUS
SYSTEM
91
INJURY IN THE NUDE MOUSE
TABLE
1
Enzyme Activities of Right and Left Sides of Different Brain Regions of Nude (Nu/Nu) and Heterozygous (Nu/+) Mice” Enzyme
activity
(&molelgih)
Right side CR) Brain
region
NuiNu
Tyrosine hydroxylase activity Midbrain ventral tegme”t”m Olfactory tubaa Striatum Frontal cortex Glutamic acid decarboxylaseactivity Midbrain ventral tegmentum n Results
0.27 0.50 0.55 0.0089
c ? 2 2
Left side (LI NuiNu
Nu/+
1.3 2.5 3.5 o.ooo9
17.3 ? 1.0
are the means e SE for groups
0.26 0.48 0.56 0.0091
? ? 2 2
1.5 2.7 3.4 o.tmo9
17.5 k I.1
0.25 0.47 0.52 0.009
* ? f 2
Ratio Nu/+
1.s 2.1 3.8 o.ooo9
17.6 2 I.2
0.24 0.50 0.54 0.0091
+ e f -+
1.7 2.5 3.7 o.ooo9
17.0 + 1.0
R/L
NuiNu
Nu/+
1.08 I.06 1.06 0.99
1.08 O.% 1.04 1.01
0.98
1.03
of SIX animals
Time Course of Changes in TH and GAD Activities after Forebrain Hemisections Changes in TH Activity. After forebrain hemisections in both the striatum (Fig. 2) and olefactory tubercle (Fig. 3), TH activity declined within 2 to 3 days to about 20% of control. Although at 42 days, TH activity in these brain regions appeared to be slightly higher in nude mice than in heterozygous mice, TH was not elevated above low activities at 2 days postoperatively, indicating that recovery did not take place. In the frontal cortex 42 days after placement of a forebrain hemisection (Fig. 4), TH activity was similarly depressed in both heterozygous and nude mice. To ascertain that its depletion in the DA terminal regions was not associated with death of parent cell bodies in the midbrain, TH activity was measured in the midbrain ventral tegmentum as well. At 42 days after the lesion no significant differences were found (Table 2). Occasionally an animal showed a reduction in TH activity in the midbrain. Such animals were excluded from the study. Changes in GAD activity. In both groups of mice the GAD activity in the midbrain ventral tegmentum region decreased to 70% of control activity within 2 days after the lesion, and remained so for as long as 42 days, indicating a lack of recovery of GABA terminals (Fig. 5).
GILAD,
92
02
GILAD,
AND KOPIN
7
42 DAYS
POSTDPERATIVE
FIG. 2. Activity of tyrosine hydroxylase (TH) in the ipsilateral striatum of nude and heterozygous mice at various times after unilateral forebrain hemisection. Results are expressed as a percentage of the contralateral side (shaded area). Each point is the mean + SE of five or six animals. At 7 days duplicate measurements were made in a single nude mouse. ** = P < 0.001 compared to the side without lesion.
02
7
30 DAYS
42
POSTOPERATIVE
FIG. 3. Activity of tyrosine hydroxylase (TH) in the ipsilateral olfactory tubercle of nude and heterozygous mice at various times after unilateral forebrain hemisection. The results are expressed as a percentage of the contralateral side (shaded area). Each point is the mean &SE value of five or six animals. At 7 days duplicate measurements were made in a single nude mouse. ** = P < 0.001 compared to the side without lesion.
CENTRAL
NERVOUS
SYSTEM
INJURY
Heterozygous
IN THE
NUDE
MOUSE
93
Nude
FIG. 4. Activity of tyrosine hydroxylase (TH) in the ipsilateral frontal cortex of heterozygous (clear bar) and nude (shaded bar) mice 42 days after unilateral forebrain hemisections. The results are expressed as a percentage of the contralateral side (shaded area). Each point is the mean + SE value of six animals. ** = P < 0.01.
Spinal Cord Hemisections Changes in Posture and Locomotion. On recovery from the surgery both nude and heterozygote mice demonstrated a flaccid paralysis of the hind limb ipsilateral to the injury and a slight contralateral bend of the torso (Fig. 6, upper panel). This motor deficit subsided within 2 to 3 days and gait and posture became apparently normal. However, ipsilateral spasticity of the hind limb could be demonstrated thereafter and did not improve for as long as 70 days after the lesion (Fig. 6, lower panel). TABLE
2
Activity” of TH in the Midbrain Ventral Tegmentum of Nude (NuiNu) and Heterozygous (Nu/+) Mice 42 Days after Forebrain Hemisectionb Group NulNu
Nu/+
Contralateral (control) side
lpsilateral (with lesion) side
250 k 13 245 + 15
235 -t 17 220 k 12
c(Enzyme activity is expressed in nanomoles [ I-W]dopa/gram b Results are the means k SE for groups of six animals.
wet weight/hour.
94
GILAD,
114
02
GILAD,
AND KOPIN
1
I
1
7
30
42
DAYS
POSTOPERATIVE
FIG. 5. Activity of glutamic acid decarboxylase (GAD) in the ipsilateral midbrain ventral tegmentum of nude and heterozygous mice at various times after unilateral forebrain hemisection. Results are expressed as a percentage of the contralateral side (shaded area). Each point is the mean 2 SE value of five or six animals. At 7 days duplicate measurements were made in a single nude mouse. ** = P < 0.01 compared to the side without lesion.
Changes in Posture and Locomotion Subsequent to a Replicate of the Lesion. To test whether or not spinal tracts regenerated and functional connections were formed, animals were subjected to a second hemisection at the same site as the first. The early ipsilateral hind limb flaccid paralysis and contralateral curved posture did not result after the second injury (Fig. 7, upper panel), indicating that animals of both groups failed to recover and were not dependent again on mechanisms similar to the ones destroyed by the original lesion. Furthermore, ipsilateral hind limb spasticity was unchanged, and was evident immediately upon recovery from surgery (Fig. 7, lower panel). DISCUSSION The results reported indicate that in the mutant nude mouse, as in the heterozygote, central neurons do not regenerate after axonal injury. After forebrain hemisection, the activities of TH and GAD rapidly decline within 2 days to a permanently depressed activity in the terminal areas of DA and GABA neurons, respectively. This anterograde reaction to the injury is similar to that previously observed in the rat (7, 19, 22), and is generally characteristic of other neuronal systems such as the NE (18) and cholinergic (13) systems. This permanent decrease in enzyme activities is a
CENTRAL
NERVOUS
SYSTEM INJURY IN THE NUDE MOUSE
95
FIG 6. Photographs of heterozygous (left side) and nude (right side) mice after left spinal cord hemisection. Upper panel: 1 day after the lesion, there is flaccid paralysis of the ipsilateral hind leg and a slight contralateral bend of the body. Lower panel: 70 days after the lesions, ipsilateral hind leg spasticity is evident.
result of a loss of enzyme molecules from degenerating terminals (18, 19), and is a direct reflection of the time course of degeneration. Degeneration appears to be permanent because enzyme activities remained depressed for as long as 42 days after the lesion. After spinal cord hemisection, a rapid recovery from the initial flaccid paralysis commenced within 2 to 3 days after the injury, and the animals appeared to have a normal gait and posture. Early recovery to this extent was documented in other mammals as well (9, IO). However, hind limb spasticity did not subside for as long as 70 days after the original lesion. Because repeated lesions at the original site did not result in any symptoms indicative of regeneration, the initial recovery probably occurred through functional compensation rather than actual structural regeneration by axonal regrowth.
FIG. 7 Photographs of heterozygous and nude mice immediately after a second spinal cord hemisection. Upper panel: both heterozygous (left side) and nude (right side) mice assume an apparently normal posture. Lower panel: ipsilateral hind leg spasticity is present in both nude (left side) and heterozygous (right side) mice.
The failure to demonstrate any evidence of axonal regeneration or any improved recovery after central nervous system lesions in the homozygous nude mouse, compared with the heterozygous trait, rules out completely the participation of the cellular part of the immune response in preventing axonal regeneration. The findings do not support the hypothesis of an immune response directed against the central nervous system to prevent regeneration (2,4,24). Forms of immunological response which remain in the mutant nude mice, even at a reduced level, were not completely ruled out because even a decreased but specific humoral immune response active locally in the lesion site, or indirectly triggering other preventive mechanisms, may be sufficient to hinder regeneration. Note Added in Proof. While this manuscript was being prepared for publication, Knowles and Berry (12) published results which led them to conclusions similar to ours.
CENTRAL
NERVOUS
SYSTEM
INJURY IN THE NUDE MOUSE
97
REFERENCES 1. BENTWICH, Z.. S. D. DOUGLAS, F. P. SIEGAL, AND H. G. KUNKEL. 1973. Human lymphocyte-sheep erythrocyte rosette formation: Some characteristics of the interaction. Clin. Immunol. Immunopathol. 1: 511-522. 2. BERRY, M., AND A. C. RICHES. 1974. An immunological approach to regeneration in the central nervous system. Br. Med. Bull. 30: 13% 140. 3. BLOEMMEN, J., AND H. EYSSEN. 1973. Immunoglobulin levels of sera of genetically thymusless (nude) mice. Eur. J. Immunol. 3: 117- 120. 4. FERINGA. E. R., J. S. WENDT, AND R. D. JOHNSON. 1974. Immunosuppressive treatment to enhance spinal cord regeneration in rats. Neurology (Minneapolis) 24: 287-293. 5. FONNUM, F.. I. GROFOVA, E. RINVIK, J. STORM-MATHISEN. AND F. WALBERG. 1974. Origin and distribution of glutamate decarboxylase in substantia nigra of the cat. Bruin Res. 71: 77-92. 6. FONNUM, F., 1. WALAAS, AND E. IVERSEN. 1977. Localization of gabaergic, cholinergic and aminergic structures in the mesolimbic system. J. Neurochem. 29: 221-230. 7. GILAD, G. M., AND D. J. REIS. 1978. Reversible reduction of tyrosine hydroxylase enzyme protein during the retrograde reaction in mesolimbic dopaminergic neurons. Brain Res. 149: 141-153. 8. GILAD, G. M.. AND D. J. REIS. 1979. Collateral sprouting in mesolimbic dopamine neurons: biochemical and immunocytochemical evidence of changes in the activity and distribution of tyrosine hydroxylase in terminal fields and in cell bodies of A 10 neurons. Brain Res. (in press). 9. GOLDBERGER. M. E. 1974. Recovery of movement following lesions ofthe motor systems in monkeys. Pages 71-100 in D. G. STERN, Ed., Recovery of Funcfion after Brain Damage. Academic Press, New York. 10. GOLDBERGER, M. E. 1977. Locomotor recovery after unilateral hindlimb deafferentation in cats. Brain Res. 123: 59-74. 11. GUTH, L., AND W. F. WINDLE. 1970. The enigma of central nervous regeneration. Exp. Neural. (Suppl). 5: l-43. 12. KNOWLES, J. F., AND M. BERRY. 1978. Effects of deoxycorticosterone acetate on regeneration of axons in the mammalian central nervous system. Exp. Neural. 62:
l-15. 13. LEWIS, P. R., C. C. D. SHUTE, acetylase analyses Physiol. (London)
14. LINDVALL. catecholamine fluorescence 15. PANTELOURIS,
0..
of a massive
AND A. SILVER. 1967. cholinergic innervation
Confirmation from choline of the rat hippocampus. J.
191: 215-224.
AND A. BJORKLUND. 1974. The organization of the ascending neuron systems in the rat brain as revealed by the glyoxylic acid method. Acta Physiol. Stand. (Suppl). 412: l-47. E. M. 1968. Absence of thymus in a mouse mutant. Nature (London) 217:
370-371. 16. PUCHALA, E., AND W. F. WINDLE. 1977. The possibility of structural and functional restitution after spinal cord injury. A review. Exp. Neurol. 55: l-42. 17. REIS. D. J., AND R. A. Ross. 1973. Dynamic changes in brain dopamine-P-hydroxylase activity during anterograde and retrograde reactions to injury of central noradrenergic neurons. Bruin Res. 57: 307-326. 18. REIS. D. J., R. A. Ross, AND T. H. JOH. 1974. Some aspects of the reaction of central and peripheral noradrenergic neurons lo injury. Pages 109- 125 in K. FUXE, L. OLSON, AND Y. ZOTTERMAN, Eds., Dynamics of Degeneration and Growth in Neurons. Pergamon Press, New York.
98
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AND KOPIN
19. REIS, D. J., G. GILAD, V. M. PICKEL, AND T. H. JOH. 1978. Reversible changes in the activities and amounts of tyrosine hydroxylase in dopamine neurons of the substantia nigra in response to axonal injury as studied by immunochemical and immunocytochemical methods. Brain Res. 144: 325-342. 20. ROBERTS, E., AND D. G. SIMONSEN. 1963. Some properties of I-glutamic acid decarboxylase in mouse brain. Biochem. Phnrmacol. 12: 113- 134. 21. RYGAARD, J. 1974. Skin grafts in “nude” mice. 3. Fate of grafts from man and other taxonomic classes. Acta Pathol. Microbial. &and. [A] 82: 105- 112. 22. STORM-MATHISEN, J. 1975. Accumulation of glutamic acid decarboxylase in the proximal parts of presumed GABA-ergic neurones after axotomy. Brain Res. 87: 107- 109. 23. UNGERSTEDT, U. 1971. Stereotaxic mapping of the monoamine pathways in the rat brain. Acta Physiol. Stand. 82 (Suppl.) 367: l-48. 24. WINDLE, W. F. 1974. Prospects of regeneration in the mammalian central nervous system. J. Sci. Lab. Denison Univ. 55: 13-19. 25. ZALEWSKI, A. A., G. F. CRESWELL, H. G. GOSHGARIAN, AND T. H. OH. 1977. The nude mouse: An in vivo model for demonstrating cross-species trophic nerve function. Exp Neural. 54: 397-402.
Reaction
NEUROLOGY
65, 87-98 (1979)
of the Mutant Mouse Nude to Axonal the Central Nervous System
GAD M. GILAD, VARDA H. GILAD,’ Laboratory
of Clinical
Science, Bethesda.
Received
National Maryland
December
of
J. KOPIN
AND IRWIN Institute 20014
Injuries
of Mental
Health,
21, 1978
The effects of unilateral forebrain and spinal cord hemisections were examined in the mutant athymic mouse nude. The activities of tyrosine hydroxylase and glutamic acid decarboxylase within the striatum and olfactory tubercle and within the midbrain ventral tegmentum, terminal fields of dopaminergic and GABA-ergic neurons, respectively, were measured to characterize the anterograde reaction of these neurons to axonal injuries. After forebrain hemisections a rapid (within 2 days) and permanent decrease was observed for all enzymes, indicating a permanent degeneration of axons with no recovery. Unilateral spinal cord hemisections resulted in a flaccid paralysis of the ipsilateral hind limb which subsided within 2 to 3 days after the lesion, and thereafter a permanent spasticity ensued. When spinal cord hemisection was repeated the initial flaccid paralysis did not occur and spasticity of the ipsilateral hind limb was immediately evident. The results indicate that regeneration of cut axons or even an improved motor recovery does not occur in the central nervous system of the nude mouse.
INTRODUCTION Regeneration of cut axons in the mammalian central nervous system is extremely limited and although some regenerative growth of cut axons occurs there, restitution of the original connections or functions has never been demonstrated. Several theories have been advanced (11, 15, 16) to explain this incapacity. One such theory, summarized recently (2), implicates immunological mechanisms: after injury, brain antigens reach the systemic circulation and result in antibody formation against brain Abbreviations: DA-dopaminergic, GABA--y-aminobutyric noradrenergic, TH-tyrosine hydroxylase, T-thymus-derived, decarboxylase. 1 Guest worker, Laboratory of Clinical Science.
acid-ergic, GAD-glutamic
NEacid
87 00 14-4886/79/070087-12$02.00/O Copyright 0 1979 by Academic Press, Inc. All rights of reproduction in any form reserved.
88
GILAD,
GILAD,
AND
KOPIN
proteins. To test this theory, we examined the reaction to axonal injury in the mutant nude mouse, which has an autosomal recessive trait resulting in a complete lack of cell-mediated and reduced humoral immunity (3, 15) and thus reduced immunoresistance (21, 25). Neurotransmitter synthesizing enzymes were used as specific markers to identify defined central neuronal systems (6,7, 17,22). After axonal injury of brain tracts known to contain catecholaminergic and GABA-ergic (GABA) fibers, changes in tyrosine hydroxylase (TH) and glutamic acid decarboxylase (GAD), specific markers for dopaminergic (DA) (5,7,8, 19) and GABA (6, 19,22) neurons, respectively, were examined in regions of brain in which these neurons terminate. In addition we studied the alterations in motor functions after spinal cord lesions. MATERIALS
AND METHODS
Animals. Experiments were conducted on outbred male and female homozygous nude (nu/nu) and heterozygous (Nu/+) mice (Sprague-Dawley, Madison, Wisconsin). Mice were housed five to a cage under germ-free conditions, fed ad libitum with sterilized standard laboratory chow and water, and maintained at 28°C with cycled lighting (on at 0900 h, off at 1700 h). Under these conditions animals appeared to be healthy and maintained normal body weight for the duration of the experiments. Characterization of Blood Lymphocytes. Lymphocytes were isolated on a Ficoll-Hypaque gradient (Winthrop Laboratories) and thymus-derived (T) lymphocytes were labeled by rosette formation with sheep erythrocytes according to the method of Bentwich et al. (1). The erythrocytes were pretreated enzymatically with neuraminidase (Bohring Diagnostics). Using this method we ascertained in a representative sample of animals the complete absence of T lymphocytes in nude mice and 30 to 40% T lymphocytes in heterozygous mice (Fig. 1). Cells were counted with a hemocytometer. Lesions. Unilateral hemisections were made in animals anesthetized with halothane (1.5% in 0,) under germ-free conditions. Brain or spinal cord was exposed by removing the bone with a dental drill. In brain, lesions were placed stereotaxically in the forebrain (1.8 mm posterior to bregma) to transect ascending DA fibers to the striatum and olfactory tubercle and noradrenergic (NE) fibers ascending to the cortex (23). In addition, such lesions transect descending GABA-producing fibers to the substantia nigra (6, 22). Brain lesions were made with a thin (co.5 mm), wide knife, made from a single-edge razor blade. In the spinal cord, lesions at the T5 level were made using a sharp segment of a double-edge razor blade mounted on a wood applicator stick.
CENTRAL
NERVOUS
SYSTEM
INJURY
IN THE
NUDE
MOUSE
89
90
GILAD,
GILAD, AND KOPIN
Groups of five or six operated animals and unoperated controls matched for age, gender, and strain were killed at various times after placement of the lesions, and the tissues were removed for biochemical analysis as described below. Assessment of Posture and Locomotion. After spinal hemisections animals were observed daily for changes in posture and locomotion. After surgery (groups of 12 animals) motion pictures of the animals were taken within 1 day and at 70 days after the lesion. Spasticity of the ipsilateral hind limb was most evident when the animal was trying to climb while being held by its tail. Changes in posture and locomotion produced by a second identical spinal cord lesion (in the original site) were examined in animals 70 days after the first lesion. Enzyme Assays. The animals were decapitated and their brains were rapidly removed, and the midbrain ventral tegmentum, olefactory tubercle, striatum, and frontal cortex were removed as previously described (7). The tissues were immediately frozen on dry ice and stored at -80°C. For assays, tissues were homogenized with a glass homogenizer in 10 vol (wt/vol) ice-cold 5 mM potassium phosphate buffer (PH 7.2) containing 0.1% Triton X-100 (voYvo1). Tyrosine dehydroxylase activity was assayed as described previously (19). Glutamic acid decarboxylase activity was assayed by the method of Roberts and Simonsen (20). RESULTS Forebrain Hemisections Regional Activities of Tyrosine Dehydroxylase (TH) and Glutamic Acid Decarboxylase (GAD) in the Brain. The regional distribution of TH activity
(Table 1) was similar to that previously reported in the rat (7), being about twice as high in the striatum and olefactory tubercle (which contain DA terminals) as in the midbrain ventral tegmentum (which contains DA cell bodies). Tyrosine dehydroxylase activity in the frontal cortex was low, consistent with the relatively low density of DA (14) and NE (23) terminals found in this region. There were no strain differences (Table 1). In this study postoperative enzyme changes in enzyme activity were compared in each animal using as controls the corresponding brain region on the contralateral side without lesion, in addition to homologous samples taken from unoperated control animals. In this manner it was ascertained that the unoperated side remained a stable point of reference. In unoperated animals there were no differences in enzyme activities between right and left sides of the brains (Table 1).
CENTRAL
NERVOUS
SYSTEM
91
INJURY IN THE NUDE MOUSE
TABLE
1
Enzyme Activities of Right and Left Sides of Different Brain Regions of Nude (Nu/Nu) and Heterozygous (Nu/+) Mice” Enzyme
activity
(&molelgih)
Right side CR) Brain
region
NuiNu
Tyrosine hydroxylase activity Midbrain ventral tegme”t”m Olfactory tubaa Striatum Frontal cortex Glutamic acid decarboxylaseactivity Midbrain ventral tegmentum n Results
0.27 0.50 0.55 0.0089
c ? 2 2
Left side (LI NuiNu
Nu/+
1.3 2.5 3.5 o.ooo9
17.3 ? 1.0
are the means e SE for groups
0.26 0.48 0.56 0.0091
? ? 2 2
1.5 2.7 3.4 o.tmo9
17.5 k I.1
0.25 0.47 0.52 0.009
* ? f 2
Ratio Nu/+
1.s 2.1 3.8 o.ooo9
17.6 2 I.2
0.24 0.50 0.54 0.0091
+ e f -+
1.7 2.5 3.7 o.ooo9
17.0 + 1.0
R/L
NuiNu
Nu/+
1.08 I.06 1.06 0.99
1.08 O.% 1.04 1.01
0.98
1.03
of SIX animals
Time Course of Changes in TH and GAD Activities after Forebrain Hemisections Changes in TH Activity. After forebrain hemisections in both the striatum (Fig. 2) and olefactory tubercle (Fig. 3), TH activity declined within 2 to 3 days to about 20% of control. Although at 42 days, TH activity in these brain regions appeared to be slightly higher in nude mice than in heterozygous mice, TH was not elevated above low activities at 2 days postoperatively, indicating that recovery did not take place. In the frontal cortex 42 days after placement of a forebrain hemisection (Fig. 4), TH activity was similarly depressed in both heterozygous and nude mice. To ascertain that its depletion in the DA terminal regions was not associated with death of parent cell bodies in the midbrain, TH activity was measured in the midbrain ventral tegmentum as well. At 42 days after the lesion no significant differences were found (Table 2). Occasionally an animal showed a reduction in TH activity in the midbrain. Such animals were excluded from the study. Changes in GAD activity. In both groups of mice the GAD activity in the midbrain ventral tegmentum region decreased to 70% of control activity within 2 days after the lesion, and remained so for as long as 42 days, indicating a lack of recovery of GABA terminals (Fig. 5).
GILAD,
92
02
GILAD,
AND KOPIN
7
42 DAYS
POSTDPERATIVE
FIG. 2. Activity of tyrosine hydroxylase (TH) in the ipsilateral striatum of nude and heterozygous mice at various times after unilateral forebrain hemisection. Results are expressed as a percentage of the contralateral side (shaded area). Each point is the mean + SE of five or six animals. At 7 days duplicate measurements were made in a single nude mouse. ** = P < 0.001 compared to the side without lesion.
02
7
30 DAYS
42
POSTOPERATIVE
FIG. 3. Activity of tyrosine hydroxylase (TH) in the ipsilateral olfactory tubercle of nude and heterozygous mice at various times after unilateral forebrain hemisection. The results are expressed as a percentage of the contralateral side (shaded area). Each point is the mean &SE value of five or six animals. At 7 days duplicate measurements were made in a single nude mouse. ** = P < 0.001 compared to the side without lesion.
CENTRAL
NERVOUS
SYSTEM
INJURY
Heterozygous
IN THE
NUDE
MOUSE
93
Nude
FIG. 4. Activity of tyrosine hydroxylase (TH) in the ipsilateral frontal cortex of heterozygous (clear bar) and nude (shaded bar) mice 42 days after unilateral forebrain hemisections. The results are expressed as a percentage of the contralateral side (shaded area). Each point is the mean + SE value of six animals. ** = P < 0.01.
Spinal Cord Hemisections Changes in Posture and Locomotion. On recovery from the surgery both nude and heterozygote mice demonstrated a flaccid paralysis of the hind limb ipsilateral to the injury and a slight contralateral bend of the torso (Fig. 6, upper panel). This motor deficit subsided within 2 to 3 days and gait and posture became apparently normal. However, ipsilateral spasticity of the hind limb could be demonstrated thereafter and did not improve for as long as 70 days after the lesion (Fig. 6, lower panel). TABLE
2
Activity” of TH in the Midbrain Ventral Tegmentum of Nude (NuiNu) and Heterozygous (Nu/+) Mice 42 Days after Forebrain Hemisectionb Group NulNu
Nu/+
Contralateral (control) side
lpsilateral (with lesion) side
250 k 13 245 + 15
235 -t 17 220 k 12
c(Enzyme activity is expressed in nanomoles [ I-W]dopa/gram b Results are the means k SE for groups of six animals.
wet weight/hour.
94
GILAD,
114
02
GILAD,
AND KOPIN
1
I
1
7
30
42
DAYS
POSTOPERATIVE
FIG. 5. Activity of glutamic acid decarboxylase (GAD) in the ipsilateral midbrain ventral tegmentum of nude and heterozygous mice at various times after unilateral forebrain hemisection. Results are expressed as a percentage of the contralateral side (shaded area). Each point is the mean 2 SE value of five or six animals. At 7 days duplicate measurements were made in a single nude mouse. ** = P < 0.01 compared to the side without lesion.
Changes in Posture and Locomotion Subsequent to a Replicate of the Lesion. To test whether or not spinal tracts regenerated and functional connections were formed, animals were subjected to a second hemisection at the same site as the first. The early ipsilateral hind limb flaccid paralysis and contralateral curved posture did not result after the second injury (Fig. 7, upper panel), indicating that animals of both groups failed to recover and were not dependent again on mechanisms similar to the ones destroyed by the original lesion. Furthermore, ipsilateral hind limb spasticity was unchanged, and was evident immediately upon recovery from surgery (Fig. 7, lower panel). DISCUSSION The results reported indicate that in the mutant nude mouse, as in the heterozygote, central neurons do not regenerate after axonal injury. After forebrain hemisection, the activities of TH and GAD rapidly decline within 2 days to a permanently depressed activity in the terminal areas of DA and GABA neurons, respectively. This anterograde reaction to the injury is similar to that previously observed in the rat (7, 19, 22), and is generally characteristic of other neuronal systems such as the NE (18) and cholinergic (13) systems. This permanent decrease in enzyme activities is a
CENTRAL
NERVOUS
SYSTEM INJURY IN THE NUDE MOUSE
95
FIG 6. Photographs of heterozygous (left side) and nude (right side) mice after left spinal cord hemisection. Upper panel: 1 day after the lesion, there is flaccid paralysis of the ipsilateral hind leg and a slight contralateral bend of the body. Lower panel: 70 days after the lesions, ipsilateral hind leg spasticity is evident.
result of a loss of enzyme molecules from degenerating terminals (18, 19), and is a direct reflection of the time course of degeneration. Degeneration appears to be permanent because enzyme activities remained depressed for as long as 42 days after the lesion. After spinal cord hemisection, a rapid recovery from the initial flaccid paralysis commenced within 2 to 3 days after the injury, and the animals appeared to have a normal gait and posture. Early recovery to this extent was documented in other mammals as well (9, IO). However, hind limb spasticity did not subside for as long as 70 days after the original lesion. Because repeated lesions at the original site did not result in any symptoms indicative of regeneration, the initial recovery probably occurred through functional compensation rather than actual structural regeneration by axonal regrowth.
FIG. 7 Photographs of heterozygous and nude mice immediately after a second spinal cord hemisection. Upper panel: both heterozygous (left side) and nude (right side) mice assume an apparently normal posture. Lower panel: ipsilateral hind leg spasticity is present in both nude (left side) and heterozygous (right side) mice.
The failure to demonstrate any evidence of axonal regeneration or any improved recovery after central nervous system lesions in the homozygous nude mouse, compared with the heterozygous trait, rules out completely the participation of the cellular part of the immune response in preventing axonal regeneration. The findings do not support the hypothesis of an immune response directed against the central nervous system to prevent regeneration (2,4,24). Forms of immunological response which remain in the mutant nude mice, even at a reduced level, were not completely ruled out because even a decreased but specific humoral immune response active locally in the lesion site, or indirectly triggering other preventive mechanisms, may be sufficient to hinder regeneration. Note Added in Proof. While this manuscript was being prepared for publication, Knowles and Berry (12) published results which led them to conclusions similar to ours.
CENTRAL
NERVOUS
SYSTEM
INJURY IN THE NUDE MOUSE
97
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