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Spinal cord injury or spinal anesthesia eliminates seizures in myelin-deficient rats
Neuroscience Letters, 84 (1988) 68 72 Elsevier Scientific Publishers Ireland l,td
68
NSL 05052
Spinal cord injury or spinal anesthesia eliminates seizures in myelin-deficient rats J. Rosenbluth and M. Hasegawa Departments of Physiology and Rehabilitation Medicine, New York University School of Medicine, New York, N Y 10016 (U.S.A,) (Received 13 July 1987; Revised version received 18 September 1987; Accepted 25 September 1987)
Key words." Myelin; Seizure; Demyelination; Spinal cord; Rat The generalized tonic seizures that occur in myelin-deficient rats can be eliminated temporarily by spinal cord injury or spinal anesthesia. These observations imply that the seizures in this mutant can be triggered by activity in the spinal cord. The results are consistent with an earlier proposal that axons in myelindeficient CNS fiber tracts can interact to produce abnormal excitation.
The myelin-deficient rat mutant develops normal myelin sheaths in the peripheral nervous system, but fiber tracts in the central nervous system remain virtually devoid of myelin [1], and what little myelin does form is abnormal [7]. Despite this deficiency, the animals survive for several weeks; however, they develop an intention tremor, primarily in the hindquarters, at ,-~2 weeks and generalized seizures at ,-~3 weeks. The seizures are tonic, characterized by extension of upper and lower extremities, hyperextension of the neck and back, temporary cessation of breathing and of the tremor, unresponsiveness to pain, and incontinence. Initially, the seizures occur only in response to vigorous stimulation, e.g. dropping the rat onto a foam pad from a height of 12 inches. Within a few days, seizures begin to appear after mild stimulation, e.g. gentle handling. Ultimately they occur spontaneously, increasing in frequency and severity with time until the animals die, usually by the end of the fourth week. The cause of the seizures is not known. There is no evidence of brain pathology, other than the myelin deficiency, and therefore it has been proposed that the seizures develop as a result of abnormal activity originating in CNS fiber tracts [6, 7]. Other myelin-deficient rodent mutants, the jimpy, shiverer and mid (myelin deficient) mice, also develop generalized seizures, and recent evidence indicates that replacement of the defective myelin basic protein gene in told partially restores myelin and reduces Correspondence." J. Rosenbluth, Depts. of Physiology & Rehabilitation Medicine, New York University School of Medicine, New York, NY 10016, U.S.A. 0304-3940/88/$ 03.50 O 1988 Elsevier Scientific Publishers Ireland Ltd.
69 seizures [4]. Focal 'paroxysmal' activity has also been reported to occur in some cases of human myelin abnormality, including multiple sclerosis patients [3]. One myelin-deficient rat maintained in our colony was observed to develop a hind limb paresis spontaneously. It was surmised that violent contraction of the paraspinal muscles during a seizure had caused a vertebral fracture resulting in a spinal cord injury. Unexpectedly, this animal survived longer than other affected animals and had a less conspicuous tremor and less frequent seizures after development of the paresis. These observations prompted a further investigation into the possibility that reduction in ascending traffic in the spinal cord of these animals might reduce their seizures. Accordingly, the following procedure was employed for producing a focal spinal cord lesion. Litters containing two or more myelin-deficient rats were identified by the characteristic intention tremor. During the third week, when seizures could be elicited, a low-thoracic laminectomy was performed on the experimental group under chloral hydrate anesthesia. After exposure of the spinal cord, 5 gl of absolute ethanol was injected into the dorsal portion of the cord on each side of the midline under direct observation. Control animals were either left untouched or subjected to laminectomy and injected with 5/11 of Ringer's solution into each half of the spinal cord. After this procedure, animals were followed for spontaneous and elicited seizure activity and tremor during walking. Sensory impairment was assessed by observing reaction to a pinch of the tail or the toes, and degree of paresis was assessed by observing walking and by the ability of the animals to climb out of an upright 100 ml beaker. All 5 ethanol-injected animals developed signs of spinal cord injury with paresis of the lower body. In some cases the animals had a diminished but still detectable sensitivity to pain and used their lower limbs in a coordinated manner during walking but were unable to escape from a beaker. In some cases the paresis was more severe, with virtual elimination of pain sensation below the level of the lesion and little residual m o t o r activity in the lower extremities. In every case, however, all manifestations of the seizures, both above and below the level of the spinal lesion, were eliminated following the procedure, and the intention tremor was also markedly diminished. Injection of the same dose of ethanol intraperitoneally, to test for possible systemic effects, had no discernable effect on either seizures or tremors. Following the intraspinal ethanol injections (day 1), seizures were not seen again in 3 of the experimental animals until days 3, 5 and 8, respectively, and were not seen at all in two other animals that were found dead on day 4. The animal that redeveloped seizures on day 3 had only a very mild residual paresis on that day and was able to escape t¥om the beaker. The results of one ethanol-injection series is shown in Table I. In one control animal that was not operated on, seizures and tremors continued. In two control animals in which Ringer's solution was injected into the spinal cord, the seizures, which had ceased during the anesthesia, returned after the anesthesia had worn offand were detectable again by the day after surgery. The tremor returned undiminished as well. Seizures in the control animals became more frequent with time, and Dilantin was administered to some twice a day, after testing them, in order
70
TABLE
I
EFFECTS
OF INTRASPINAL
ETHANOL
A f t e r d a y 1, a n i m a l s w e r e t e s t e d t w i c e e a c h d a y ( a . m , a n d p . m . ) a n d t h e r e s u l t s r e c o r d e d + or
. Procedures performed are indicated
by letters.
Dilantin
was
as + + + , + ~ .
to a n i m a l 3 o n da3
administered
4 ( p . m . ) a n d to a n i m a l 4 o n d a y 3 ( p . m . ) a n d d a y 4 ( a . m . a n d p . m . ) . L, l a m i n e c t o m y ; E, i n t r a s p i n a l e t h a n o l : R, i n t r a s p i n a l R i n g e r ' s s o l u t i o n . Seizure Animal
1
2
Tremor 3
4
I
Paresis 2
3
4
+++
+++
1
2
3
4
Day I a.m.
+
+
LE
LE
-
+++
+++
LE
LE
.
.
.
+
+
+++
+
LR +++
+++
+
+
LE
LE
+
+
.
.
Day 2 a.m.
. . . .
+
-
LR p. hi.
-
+
+
+
+++
-
_
LR
Day 3 a.m.
_
_
+
+
+
+++
+++
+
+
--
p.m.
_
_
+
+
+
+
+++
+++
+
+
.....
a.m.
_
_
+
+
+
+
+++
+++
+
+
-
p.m.
.....
+
+
+
+
+++
+++
+
+
--
+
+
+
+
+++
+++
+
+
_
Day 4 _
Day 5 a.m.
+
--
E p.m.
-
E -
E +
+
E
E
-
+
E +
+
to reduce the frequency of the seizures and prolong their lives. This proved to be of only limited value, however. In contrast, intraspinal injection of ethanol into one control animal on day 5 eliminated the seizures. In another group of 7 animals, laminectomy was performed and the skin closed over the defect. The following day the animals were injected intraspinally at the laminectomy site with 0.15 ml of either 2% procaine or Ringer's solution. In some animals the procedure was repeated one or more times. In all of 14 trials with procaine, the animals displayed a marked diminution in their tremor within ~ 5 min (mean, 4.6 min; range 1-10 min). They developed obvious paresis at approximately the same time, and seizures could no longer be elicited. These effects lasted approximately 1-2 h. The paresis then gradually subsided, tremors returned and, at later times, seizures could once again be elicited or appeared spontaneously. Intraspinat injection of Ringer's solution had no effect on tremor or seizures and did not cause paresis. The result of one procaine-injection series is shown in Table II. It is concluded from these studies that generalized seizures in myelin-deficient rats can be triggered by activity in the spinal cord. Otherwise, spinal anesthesia or spinal
71
TABLE II E F F E C T S OF I N T R A S P I N A L P R O C A I N E Test results and procedures are recorded as in Table I. L, laminectomy: P, intraspinal procaine: R, intraspinal Ringer's solution. Seizure Animal Pre-op.
1
Tremor 2
3
1
Paresis 2
3
+
+
+
+++
+++
+++
L
L
L
L
L
L
1
2
3
L
L
t.
P
R
Pre-inj.
+
+
+
+++
+++
+++
0 n]ln
P
P
R +
P
P
R +++
P +
_
_
+
+ + +
+
-
+
-
_
+ + +
+
+
-
+ + +
+
+
+
+++
+
+
+ + +
2 min 5 min I0 mm 15 min 20 min 50 min 60 rain
_
-
_
+ + +
-
cord injury would not prevent their occurrence, but would only reduce their manifestations in the lower body and hindlimbs. The characteristic hyperextension of the neck, extension of the upper extremities, cessation of breathing and loss of response to pain that occur during seizures would all persist. The fact that the seizures are eliminated temporarily by spinal cord injury or spinal anesthesia implies that activity below the level of the block contributes significantly to the initiation of seizures in these animals. It has been demonstrated previously that in both normal and myelin-deficient rats there is a marked increase in voltage-sensitive sodium channels in the CNS between 6 and 21 days after birth [2] and that patches similar to nodes of Ranvier [5] occur in the myelin-deficient axons. If these patches represent aggregates of sodium channels [6], they would constitute regions of increased ion exchange and reduced threshold for excitation. Such sites could thus underlie spontaneous activity in the axons, during transient elevation of extracellular potassium levels associated with activity in adjacent axons, for example [6]. In addition, the node-like patches may also be regions of reduced transmembrane resistance, in view of the surprisingly low resistance of the axolemma at normal nodes of Ranvier [8], and could thus represent sites of invasion by currents generated in neighboring active axons. In either case synchronized, spontaneous activity could be generated in otherwise silent axons, and the traffic of impulses ascending in fiber tracts correspondingly increased. Since spinal cord injury or anesthesia blocks ascending pathways, it can be expected to reduce the amount of this traffic reaching higher centers. The redevelopment of seizures in the spinal cord-injured animals, as they get older, may reflect a continuing increase in the concentration of ion channels in fiber tracts
72 above the level of the injury, as well as in u n d a m a g e d fiber tracts below the level of the injury, ultimately resulting in sufficient a b n o r m a l activity to trigger seizures once again. In s u m m a r y , partial destruction or a n e s t h e t i z a t i o n of the thoracic spinal cord in myelin-deficient rats results in a t e m p o r a r y e l i m i n a t i o n o f the generalized tonic seizures characteristic o f this m u t a n t . The o b s e r v a t i o n s s u p p o r t the proposal that the seizures can be triggered by a b n o r m a l activity o r i g i n a t i n g in myelin-deficient fiber tracts of the spinal cord a n d can be prevented by blocking c o n d u c t i o n of this activity to higher centers. S u p p o r t e d by grants from the N I H (NS 07495) a n d N a t i o n a l Multiple Sclerosis Society ( R G 1579). 1 Dentinger, M.P., Barron, K.D. and Csiza, C.K., Uttrastructure of the CNS in a myelin-deficientrat, J. Neurocytol., 11 (1982)671-691. 20aklander, A.L., Pellegrino, R. and Ritchie, J.M., Saxitoxin binding to central and peripheral nervous tissue of the myelin deficiency(rod) mutant rat, Brain Res., 307 (1984) 393-397. 30sterman, P.O. and Westerberg, C.E., Paroxysmal attacks in multiple sclerosis, Brain, 98 (1975) 189 202. 4 Popko, B., Puckett, C., Lai, E., Shine, H.D., Readhead, C., Takahashi, N., Hunt III, S.W., Sidman, R.L. and Hood, L., Myelin deficient mice: expression of myelin basic protein and generation of mice with varying levels of myelin, Cell, 48 (1987) 713-721. 5 Rosenbluth, J., Membrane specializations at the nodes of Ranvier, paranodal and juxtaparanodal regions of myelinated central and peripheral nerve fibers. In J. Zagoren and S. Fedoroff (Eds.), The Node of Ranvier, Vol. 1, Cellular Neurobiology Academic, New York, 1984, pp. 31-67. 6 Rosenbluth, J., Intramembranous particle patches in myelin-deficient rat axons, Neurosci. Lett., 62 (1985) 19 24. 7 Rosenbluth, J., Abnormal axoglial junctions in the myelin-deficient rat mutant, J. Neurocytol., 16 (1987) 497- 509. 8 Tasaki, 1., New measurements of the capacity and resistance of the myelin sheath and the nodal membrane of the isolated frog nerve fiber, Am. J. Physiol., 181 (1955) 639-650.
68
NSL 05052
Spinal cord injury or spinal anesthesia eliminates seizures in myelin-deficient rats J. Rosenbluth and M. Hasegawa Departments of Physiology and Rehabilitation Medicine, New York University School of Medicine, New York, N Y 10016 (U.S.A,) (Received 13 July 1987; Revised version received 18 September 1987; Accepted 25 September 1987)
Key words." Myelin; Seizure; Demyelination; Spinal cord; Rat The generalized tonic seizures that occur in myelin-deficient rats can be eliminated temporarily by spinal cord injury or spinal anesthesia. These observations imply that the seizures in this mutant can be triggered by activity in the spinal cord. The results are consistent with an earlier proposal that axons in myelindeficient CNS fiber tracts can interact to produce abnormal excitation.
The myelin-deficient rat mutant develops normal myelin sheaths in the peripheral nervous system, but fiber tracts in the central nervous system remain virtually devoid of myelin [1], and what little myelin does form is abnormal [7]. Despite this deficiency, the animals survive for several weeks; however, they develop an intention tremor, primarily in the hindquarters, at ,-~2 weeks and generalized seizures at ,-~3 weeks. The seizures are tonic, characterized by extension of upper and lower extremities, hyperextension of the neck and back, temporary cessation of breathing and of the tremor, unresponsiveness to pain, and incontinence. Initially, the seizures occur only in response to vigorous stimulation, e.g. dropping the rat onto a foam pad from a height of 12 inches. Within a few days, seizures begin to appear after mild stimulation, e.g. gentle handling. Ultimately they occur spontaneously, increasing in frequency and severity with time until the animals die, usually by the end of the fourth week. The cause of the seizures is not known. There is no evidence of brain pathology, other than the myelin deficiency, and therefore it has been proposed that the seizures develop as a result of abnormal activity originating in CNS fiber tracts [6, 7]. Other myelin-deficient rodent mutants, the jimpy, shiverer and mid (myelin deficient) mice, also develop generalized seizures, and recent evidence indicates that replacement of the defective myelin basic protein gene in told partially restores myelin and reduces Correspondence." J. Rosenbluth, Depts. of Physiology & Rehabilitation Medicine, New York University School of Medicine, New York, NY 10016, U.S.A. 0304-3940/88/$ 03.50 O 1988 Elsevier Scientific Publishers Ireland Ltd.
69 seizures [4]. Focal 'paroxysmal' activity has also been reported to occur in some cases of human myelin abnormality, including multiple sclerosis patients [3]. One myelin-deficient rat maintained in our colony was observed to develop a hind limb paresis spontaneously. It was surmised that violent contraction of the paraspinal muscles during a seizure had caused a vertebral fracture resulting in a spinal cord injury. Unexpectedly, this animal survived longer than other affected animals and had a less conspicuous tremor and less frequent seizures after development of the paresis. These observations prompted a further investigation into the possibility that reduction in ascending traffic in the spinal cord of these animals might reduce their seizures. Accordingly, the following procedure was employed for producing a focal spinal cord lesion. Litters containing two or more myelin-deficient rats were identified by the characteristic intention tremor. During the third week, when seizures could be elicited, a low-thoracic laminectomy was performed on the experimental group under chloral hydrate anesthesia. After exposure of the spinal cord, 5 gl of absolute ethanol was injected into the dorsal portion of the cord on each side of the midline under direct observation. Control animals were either left untouched or subjected to laminectomy and injected with 5/11 of Ringer's solution into each half of the spinal cord. After this procedure, animals were followed for spontaneous and elicited seizure activity and tremor during walking. Sensory impairment was assessed by observing reaction to a pinch of the tail or the toes, and degree of paresis was assessed by observing walking and by the ability of the animals to climb out of an upright 100 ml beaker. All 5 ethanol-injected animals developed signs of spinal cord injury with paresis of the lower body. In some cases the animals had a diminished but still detectable sensitivity to pain and used their lower limbs in a coordinated manner during walking but were unable to escape from a beaker. In some cases the paresis was more severe, with virtual elimination of pain sensation below the level of the lesion and little residual m o t o r activity in the lower extremities. In every case, however, all manifestations of the seizures, both above and below the level of the spinal lesion, were eliminated following the procedure, and the intention tremor was also markedly diminished. Injection of the same dose of ethanol intraperitoneally, to test for possible systemic effects, had no discernable effect on either seizures or tremors. Following the intraspinal ethanol injections (day 1), seizures were not seen again in 3 of the experimental animals until days 3, 5 and 8, respectively, and were not seen at all in two other animals that were found dead on day 4. The animal that redeveloped seizures on day 3 had only a very mild residual paresis on that day and was able to escape t¥om the beaker. The results of one ethanol-injection series is shown in Table I. In one control animal that was not operated on, seizures and tremors continued. In two control animals in which Ringer's solution was injected into the spinal cord, the seizures, which had ceased during the anesthesia, returned after the anesthesia had worn offand were detectable again by the day after surgery. The tremor returned undiminished as well. Seizures in the control animals became more frequent with time, and Dilantin was administered to some twice a day, after testing them, in order
70
TABLE
I
EFFECTS
OF INTRASPINAL
ETHANOL
A f t e r d a y 1, a n i m a l s w e r e t e s t e d t w i c e e a c h d a y ( a . m , a n d p . m . ) a n d t h e r e s u l t s r e c o r d e d + or
. Procedures performed are indicated
by letters.
Dilantin
was
as + + + , + ~ .
to a n i m a l 3 o n da3
administered
4 ( p . m . ) a n d to a n i m a l 4 o n d a y 3 ( p . m . ) a n d d a y 4 ( a . m . a n d p . m . ) . L, l a m i n e c t o m y ; E, i n t r a s p i n a l e t h a n o l : R, i n t r a s p i n a l R i n g e r ' s s o l u t i o n . Seizure Animal
1
2
Tremor 3
4
I
Paresis 2
3
4
+++
+++
1
2
3
4
Day I a.m.
+
+
LE
LE
-
+++
+++
LE
LE
.
.
.
+
+
+++
+
LR +++
+++
+
+
LE
LE
+
+
.
.
Day 2 a.m.
. . . .
+
-
LR p. hi.
-
+
+
+
+++
-
_
LR
Day 3 a.m.
_
_
+
+
+
+++
+++
+
+
--
p.m.
_
_
+
+
+
+
+++
+++
+
+
.....
a.m.
_
_
+
+
+
+
+++
+++
+
+
-
p.m.
.....
+
+
+
+
+++
+++
+
+
--
+
+
+
+
+++
+++
+
+
_
Day 4 _
Day 5 a.m.
+
--
E p.m.
-
E -
E +
+
E
E
-
+
E +
+
to reduce the frequency of the seizures and prolong their lives. This proved to be of only limited value, however. In contrast, intraspinal injection of ethanol into one control animal on day 5 eliminated the seizures. In another group of 7 animals, laminectomy was performed and the skin closed over the defect. The following day the animals were injected intraspinally at the laminectomy site with 0.15 ml of either 2% procaine or Ringer's solution. In some animals the procedure was repeated one or more times. In all of 14 trials with procaine, the animals displayed a marked diminution in their tremor within ~ 5 min (mean, 4.6 min; range 1-10 min). They developed obvious paresis at approximately the same time, and seizures could no longer be elicited. These effects lasted approximately 1-2 h. The paresis then gradually subsided, tremors returned and, at later times, seizures could once again be elicited or appeared spontaneously. Intraspinat injection of Ringer's solution had no effect on tremor or seizures and did not cause paresis. The result of one procaine-injection series is shown in Table II. It is concluded from these studies that generalized seizures in myelin-deficient rats can be triggered by activity in the spinal cord. Otherwise, spinal anesthesia or spinal
71
TABLE II E F F E C T S OF I N T R A S P I N A L P R O C A I N E Test results and procedures are recorded as in Table I. L, laminectomy: P, intraspinal procaine: R, intraspinal Ringer's solution. Seizure Animal Pre-op.
1
Tremor 2
3
1
Paresis 2
3
+
+
+
+++
+++
+++
L
L
L
L
L
L
1
2
3
L
L
t.
P
R
Pre-inj.
+
+
+
+++
+++
+++
0 n]ln
P
P
R +
P
P
R +++
P +
_
_
+
+ + +
+
-
+
-
_
+ + +
+
+
-
+ + +
+
+
+
+++
+
+
+ + +
2 min 5 min I0 mm 15 min 20 min 50 min 60 rain
_
-
_
+ + +
-
cord injury would not prevent their occurrence, but would only reduce their manifestations in the lower body and hindlimbs. The characteristic hyperextension of the neck, extension of the upper extremities, cessation of breathing and loss of response to pain that occur during seizures would all persist. The fact that the seizures are eliminated temporarily by spinal cord injury or spinal anesthesia implies that activity below the level of the block contributes significantly to the initiation of seizures in these animals. It has been demonstrated previously that in both normal and myelin-deficient rats there is a marked increase in voltage-sensitive sodium channels in the CNS between 6 and 21 days after birth [2] and that patches similar to nodes of Ranvier [5] occur in the myelin-deficient axons. If these patches represent aggregates of sodium channels [6], they would constitute regions of increased ion exchange and reduced threshold for excitation. Such sites could thus underlie spontaneous activity in the axons, during transient elevation of extracellular potassium levels associated with activity in adjacent axons, for example [6]. In addition, the node-like patches may also be regions of reduced transmembrane resistance, in view of the surprisingly low resistance of the axolemma at normal nodes of Ranvier [8], and could thus represent sites of invasion by currents generated in neighboring active axons. In either case synchronized, spontaneous activity could be generated in otherwise silent axons, and the traffic of impulses ascending in fiber tracts correspondingly increased. Since spinal cord injury or anesthesia blocks ascending pathways, it can be expected to reduce the amount of this traffic reaching higher centers. The redevelopment of seizures in the spinal cord-injured animals, as they get older, may reflect a continuing increase in the concentration of ion channels in fiber tracts
72 above the level of the injury, as well as in u n d a m a g e d fiber tracts below the level of the injury, ultimately resulting in sufficient a b n o r m a l activity to trigger seizures once again. In s u m m a r y , partial destruction or a n e s t h e t i z a t i o n of the thoracic spinal cord in myelin-deficient rats results in a t e m p o r a r y e l i m i n a t i o n o f the generalized tonic seizures characteristic o f this m u t a n t . The o b s e r v a t i o n s s u p p o r t the proposal that the seizures can be triggered by a b n o r m a l activity o r i g i n a t i n g in myelin-deficient fiber tracts of the spinal cord a n d can be prevented by blocking c o n d u c t i o n of this activity to higher centers. S u p p o r t e d by grants from the N I H (NS 07495) a n d N a t i o n a l Multiple Sclerosis Society ( R G 1579). 1 Dentinger, M.P., Barron, K.D. and Csiza, C.K., Uttrastructure of the CNS in a myelin-deficientrat, J. Neurocytol., 11 (1982)671-691. 20aklander, A.L., Pellegrino, R. and Ritchie, J.M., Saxitoxin binding to central and peripheral nervous tissue of the myelin deficiency(rod) mutant rat, Brain Res., 307 (1984) 393-397. 30sterman, P.O. and Westerberg, C.E., Paroxysmal attacks in multiple sclerosis, Brain, 98 (1975) 189 202. 4 Popko, B., Puckett, C., Lai, E., Shine, H.D., Readhead, C., Takahashi, N., Hunt III, S.W., Sidman, R.L. and Hood, L., Myelin deficient mice: expression of myelin basic protein and generation of mice with varying levels of myelin, Cell, 48 (1987) 713-721. 5 Rosenbluth, J., Membrane specializations at the nodes of Ranvier, paranodal and juxtaparanodal regions of myelinated central and peripheral nerve fibers. In J. Zagoren and S. Fedoroff (Eds.), The Node of Ranvier, Vol. 1, Cellular Neurobiology Academic, New York, 1984, pp. 31-67. 6 Rosenbluth, J., Intramembranous particle patches in myelin-deficient rat axons, Neurosci. Lett., 62 (1985) 19 24. 7 Rosenbluth, J., Abnormal axoglial junctions in the myelin-deficient rat mutant, J. Neurocytol., 16 (1987) 497- 509. 8 Tasaki, 1., New measurements of the capacity and resistance of the myelin sheath and the nodal membrane of the isolated frog nerve fiber, Am. J. Physiol., 181 (1955) 639-650.