Journal of Neuroscience Research 27:219-227 (1990)
Prolonged Ongoing Discharges of Sensory Nerves as Recorded in Isolated Nerves in the Rat E. J. Baik-Han, K.J. Kim, and J.M. Chung Marine Biomedical Institute (E.J.B.-K., K.J.K., J.M.C.), Dcpartrnents of Anatomy a n d Ncuroscienccs (J.M.C.), and Physiology and Biophysics (J.M.C.). University of Texas Medical Brarich, Galveston, Texas
Whether or not injury to a mammalian sensory nerve produces prolonged discharges is a controversial issue. Because of this controversy and its potential ramifications in both experimental and clinical conditions, we examined discharges in sectioned sensory nerves of the rat. In anesthetized rats, either a dorsal root or a saphenous nerve was isolated by sectioning both proximally and distally. Multi-unit recordings from these isolated nerves showed low levels of prolonged ongoing discharges often lasting for at least 2 hr. Furthermore, results from short-term (1-2 days) survival surgeries indicated that prolonged ongoing discharges could last for days. Sectioning the ventral root produced discharges for only a short period. Various pieces of evidence suggested that the sources of impulse generation are multiple, occurring in the middle of an uninjured axon at a site away from the injury, as well as at the injured site. There is circumstantial evidence which suggests that these prolonged discharges are produced in physiological conditions or at least under normal experimental conditions. Key words: peripheral nerve injury, dorsal rhizotomy, spontaneous activity, abnormal impulse generation, ectopic impulses, injury discharges
Despite the remarkably detailed description of POD by Adrian and its obvious importance, little attcntion has been paid to this phenomenon. For this reason, Wall and his co-workers (Wall et al., 1974) attempted to reproduce the POD as described by Adrian using cats and rats. They found that an injury to a nerve produced a burst of discharges that only lasted a few minutes. They reported that no POD could be produced in either sensory or motor nerve fibers unless the cut end of the nerve was allowed to dehydrate, the calcium level of the damaged axons was severely reduced, or sensory endings were left intact. They offered as an explanation of Adrian's observations that small side branches of the damaged nerve were likely left intact. It is important to determine whether or not injury to a sensory nerve fiber produces prolonged discharges because such a phenomenon would have significant implications in both clinical and experimental situations. Since the two previously published results on this topic contradict one another, we decided to repeat the experinient, with some modifications, using rats. Preliminary results have been published in an abstract (Han et a]., 1989).
METHODS INTRODUC'IXON Although the middle of an axon is an unusual site of impulse generation, ectopic impulses are often generated from the axon upon injury. In a classical and detailed study, Adrian ( 1930) recorded prolonged ongoing discharges (POD) from a dissected nerve trunk i n a mammalian nerve. Scction of the nerve led to the development of prolonged discharges from the cut ends of small diametcr sensory fibers. In contrast, motor fibers produced discharges only briefly after injury. Adrian categorized the POD into 3 types of patterns: 1) regular high frequency, 2) irregular low frequency, and 3) bursts. The POD of sensory nerve fibers have obvious important implications for both clinical and experimental situations. L
0 1990 Wiley-ldss, lnc.
Experiments were performed on a total of 21 adult (250-350 g) Sprague-Dawley rats of either sex. After anesthesia with sodium pentobarbital (Nembutal, 50 mgi kg, i.p.) and a tracheostomy, each rat was ventilated while maintaining an end-tidal expired CO, level bctween 3.5 and 4.5%. The lcvel of anesthesia was maintained by infusing sodium pentobarbital ( 1 0 mglkgihr) through a cannula in the external jugular vein. In most experiments, animals were paralyzed with an injection of
Keceivcd March 2, 1990; revised May 8. 1990. Address reprint requesta to J i n h1.10Chung. Ph.D., Marine Biomedical Institute. University of Texas Medical Branch, 200 University Boulevard, Galveston, Texas 77550.
220
Raik-Han et al.
gallamine triethiodide (Flaxedil, 10 mgikglhr), but some experiments were done without injection of a ncuromuscular blocking agent. The rectal temperature of the animals was maintained at 37.5"C throughout the experiment by a servo-controlled electrical heating pad. A laminectomy was performed to expose the lumbar spinal cord and spinal roots of the L4-L6 segments. The animal was mounted on a stereotaxic frame and skin flaps retracted to form a pool around the exposed spinal cord, which was then fillcd with warm mineral oil. The temperature of the oil pool was maintained at 36°C by a heating coil with circulating water. To record from a spinal root, the dura mater was opened and a root (either L4. LS, or L6) was cut and placed on a recording electrode assembly consisting of 4 platinum wires (28 gauge), each separated by 2 mm. To record from the saphenous nerve. about 3 cm of the nerve was carefully dissected out after an incision was made from the inguinal region to the medial ankle. The dissected nerve was then moved to the spinal oil pool for the actual recording. The rccorded extracellular niultiunit activity was amplified and displayed on a digital oscilloscope (Nicolct, 4094). The activity was also fed into a window discriminator whose output was used t o compile time histograms with a data acquisition systcm (CED- 1401).
RESULTS Extracellular multi-unit activity was recorded from whole nerve preparations of 86 dorsal roots, 28 ventral roots, and 3 saphenous nerves from 21 rats.
Prolonged Ongoing Discharges in isolated Dorsal Roots Under Normal Experimental Conditions Prolonged ongoing discharges (POD) were often recorded from completely isolated dorsal roots. Figure 1 shows the results of one such experiment. The distal stump of an L6 dorsal root, cut close to the spinal cord, was placed on the recording electrode assembly and then recording began. As shown in Figure l A , numerous spike potentials of diffcrcnt sizes and shapes appeared. Many of these spikes persisted after cutting the I,6 spinal nerve to eliminate most of the activity originating from pcripherdi receptors. The remaining activity could be enhanced by applying mechanical stimulation to the dorsal root ganglion (Fig. IB). As shown in Figure 1C,D, some level of activity persisted for a long time, even after cutting the root between the recording site and the dorsal root ganglion. At this time, a piece of completely isolated dorsal root lay on the recording electrode assembly in the mineral oil pool, and yet POD were still present. The presence of POD was surveyed in 55 isolated dorsal roots. Among these, various levels of POD lasting
Fig. I . Prolonged discharges recorded from an isolated dorsal root. A-D show single-pass time histograms of multi-unit activity recorded from the L6 dorsal root during various manipulations. Insets show actual recorded multi-unit action potentials in each case. A: The dorsal root was cut close to the spinal cord and unit activity was recorded from the distal stump. At the time indicated by the arrow, the Lh spinal nerve was cut just distal to the dorsal root ganglion. R: Recording began 35 min after the spinal nerve was cut. During the period of the bar, gentlc pressure was applied to the dorsal root ganglion with a blunt glass rod. C: Recording began 42 inin after the spinal nerve was cut. At the time indicated by the arrow, the dorsal root was cut just proximal to the dorsal root ganglion, isolating the root cornpletcly. D: Rccording began 95 min after complctc isolation of the root. longer than 30 min after acute isolation were observed in 25 roots. The activity often lasted for 90 inin, and the maximum length of time that we observed was 400 min. However, none of the 28 ventral roots (examined in the same way as the dorsal roots) displayed such activity longer than 15 min. Various factors that could conceivably be correlated with the duration of these dorsal root POD were investigated. Neither the side (left or right) nor the segmental level of the recorded root was correlated with thc
Prolonged Ongoing Discharges 7,
,
221
A
1
U
5 ms
. 0
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2
.
3
. 4
.
5
.
.
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,
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,
,
1 0 1 1
Distal
Sampling Time (hr)
Fig. 2 . Relationship between the duration o f prolonged injury discharges and the sampling time. The maximum duration of prolonged injury discharges recorded from each dorsal root (n = 5 5 ) was plotted against the sampling time. The sampling time is exprcsscd as hours after the first recording session. For examplc, dorsal roots belonging to sampling times 0 and 5 would be the first recorded dorsal root for a given experiment and the one recorded starting 5 hours thereafter, respectively. Correlation was highly significant (two-tailed, P
Prolonged Ongoing Discharges of Sensory Nerves as Recorded in Isolated Nerves in the Rat E. J. Baik-Han, K.J. Kim, and J.M. Chung Marine Biomedical Institute (E.J.B.-K., K.J.K., J.M.C.), Dcpartrnents of Anatomy a n d Ncuroscienccs (J.M.C.), and Physiology and Biophysics (J.M.C.). University of Texas Medical Brarich, Galveston, Texas
Whether or not injury to a mammalian sensory nerve produces prolonged discharges is a controversial issue. Because of this controversy and its potential ramifications in both experimental and clinical conditions, we examined discharges in sectioned sensory nerves of the rat. In anesthetized rats, either a dorsal root or a saphenous nerve was isolated by sectioning both proximally and distally. Multi-unit recordings from these isolated nerves showed low levels of prolonged ongoing discharges often lasting for at least 2 hr. Furthermore, results from short-term (1-2 days) survival surgeries indicated that prolonged ongoing discharges could last for days. Sectioning the ventral root produced discharges for only a short period. Various pieces of evidence suggested that the sources of impulse generation are multiple, occurring in the middle of an uninjured axon at a site away from the injury, as well as at the injured site. There is circumstantial evidence which suggests that these prolonged discharges are produced in physiological conditions or at least under normal experimental conditions. Key words: peripheral nerve injury, dorsal rhizotomy, spontaneous activity, abnormal impulse generation, ectopic impulses, injury discharges
Despite the remarkably detailed description of POD by Adrian and its obvious importance, little attcntion has been paid to this phenomenon. For this reason, Wall and his co-workers (Wall et al., 1974) attempted to reproduce the POD as described by Adrian using cats and rats. They found that an injury to a nerve produced a burst of discharges that only lasted a few minutes. They reported that no POD could be produced in either sensory or motor nerve fibers unless the cut end of the nerve was allowed to dehydrate, the calcium level of the damaged axons was severely reduced, or sensory endings were left intact. They offered as an explanation of Adrian's observations that small side branches of the damaged nerve were likely left intact. It is important to determine whether or not injury to a sensory nerve fiber produces prolonged discharges because such a phenomenon would have significant implications in both clinical and experimental situations. Since the two previously published results on this topic contradict one another, we decided to repeat the experinient, with some modifications, using rats. Preliminary results have been published in an abstract (Han et a]., 1989).
METHODS INTRODUC'IXON Although the middle of an axon is an unusual site of impulse generation, ectopic impulses are often generated from the axon upon injury. In a classical and detailed study, Adrian ( 1930) recorded prolonged ongoing discharges (POD) from a dissected nerve trunk i n a mammalian nerve. Scction of the nerve led to the development of prolonged discharges from the cut ends of small diametcr sensory fibers. In contrast, motor fibers produced discharges only briefly after injury. Adrian categorized the POD into 3 types of patterns: 1) regular high frequency, 2) irregular low frequency, and 3) bursts. The POD of sensory nerve fibers have obvious important implications for both clinical and experimental situations. L
0 1990 Wiley-ldss, lnc.
Experiments were performed on a total of 21 adult (250-350 g) Sprague-Dawley rats of either sex. After anesthesia with sodium pentobarbital (Nembutal, 50 mgi kg, i.p.) and a tracheostomy, each rat was ventilated while maintaining an end-tidal expired CO, level bctween 3.5 and 4.5%. The lcvel of anesthesia was maintained by infusing sodium pentobarbital ( 1 0 mglkgihr) through a cannula in the external jugular vein. In most experiments, animals were paralyzed with an injection of
Keceivcd March 2, 1990; revised May 8. 1990. Address reprint requesta to J i n h1.10Chung. Ph.D., Marine Biomedical Institute. University of Texas Medical Branch, 200 University Boulevard, Galveston, Texas 77550.
220
Raik-Han et al.
gallamine triethiodide (Flaxedil, 10 mgikglhr), but some experiments were done without injection of a ncuromuscular blocking agent. The rectal temperature of the animals was maintained at 37.5"C throughout the experiment by a servo-controlled electrical heating pad. A laminectomy was performed to expose the lumbar spinal cord and spinal roots of the L4-L6 segments. The animal was mounted on a stereotaxic frame and skin flaps retracted to form a pool around the exposed spinal cord, which was then fillcd with warm mineral oil. The temperature of the oil pool was maintained at 36°C by a heating coil with circulating water. To record from a spinal root, the dura mater was opened and a root (either L4. LS, or L6) was cut and placed on a recording electrode assembly consisting of 4 platinum wires (28 gauge), each separated by 2 mm. To record from the saphenous nerve. about 3 cm of the nerve was carefully dissected out after an incision was made from the inguinal region to the medial ankle. The dissected nerve was then moved to the spinal oil pool for the actual recording. The rccorded extracellular niultiunit activity was amplified and displayed on a digital oscilloscope (Nicolct, 4094). The activity was also fed into a window discriminator whose output was used t o compile time histograms with a data acquisition systcm (CED- 1401).
RESULTS Extracellular multi-unit activity was recorded from whole nerve preparations of 86 dorsal roots, 28 ventral roots, and 3 saphenous nerves from 21 rats.
Prolonged Ongoing Discharges in isolated Dorsal Roots Under Normal Experimental Conditions Prolonged ongoing discharges (POD) were often recorded from completely isolated dorsal roots. Figure 1 shows the results of one such experiment. The distal stump of an L6 dorsal root, cut close to the spinal cord, was placed on the recording electrode assembly and then recording began. As shown in Figure l A , numerous spike potentials of diffcrcnt sizes and shapes appeared. Many of these spikes persisted after cutting the I,6 spinal nerve to eliminate most of the activity originating from pcripherdi receptors. The remaining activity could be enhanced by applying mechanical stimulation to the dorsal root ganglion (Fig. IB). As shown in Figure 1C,D, some level of activity persisted for a long time, even after cutting the root between the recording site and the dorsal root ganglion. At this time, a piece of completely isolated dorsal root lay on the recording electrode assembly in the mineral oil pool, and yet POD were still present. The presence of POD was surveyed in 55 isolated dorsal roots. Among these, various levels of POD lasting
Fig. I . Prolonged discharges recorded from an isolated dorsal root. A-D show single-pass time histograms of multi-unit activity recorded from the L6 dorsal root during various manipulations. Insets show actual recorded multi-unit action potentials in each case. A: The dorsal root was cut close to the spinal cord and unit activity was recorded from the distal stump. At the time indicated by the arrow, the Lh spinal nerve was cut just distal to the dorsal root ganglion. R: Recording began 35 min after the spinal nerve was cut. During the period of the bar, gentlc pressure was applied to the dorsal root ganglion with a blunt glass rod. C: Recording began 42 inin after the spinal nerve was cut. At the time indicated by the arrow, the dorsal root was cut just proximal to the dorsal root ganglion, isolating the root cornpletcly. D: Rccording began 95 min after complctc isolation of the root. longer than 30 min after acute isolation were observed in 25 roots. The activity often lasted for 90 inin, and the maximum length of time that we observed was 400 min. However, none of the 28 ventral roots (examined in the same way as the dorsal roots) displayed such activity longer than 15 min. Various factors that could conceivably be correlated with the duration of these dorsal root POD were investigated. Neither the side (left or right) nor the segmental level of the recorded root was correlated with thc
Prolonged Ongoing Discharges 7,
,
221
A
1
U
5 ms
. 0
1
2
.
3
. 4
.
5
.
.
6
7
,
8
9
,
,
1 0 1 1
Distal
Sampling Time (hr)
Fig. 2 . Relationship between the duration o f prolonged injury discharges and the sampling time. The maximum duration of prolonged injury discharges recorded from each dorsal root (n = 5 5 ) was plotted against the sampling time. The sampling time is exprcsscd as hours after the first recording session. For examplc, dorsal roots belonging to sampling times 0 and 5 would be the first recorded dorsal root for a given experiment and the one recorded starting 5 hours thereafter, respectively. Correlation was highly significant (two-tailed, P
