Effects of Q-switched Nd:YAG laser irradiation on neural impulse propagation: I. Spinal cord.

We studied the effect of irradiation with Q-switched Nd:YAG laser light (1064 nm) on spinal cord dorsal column and dorsolateral white matter in anesthetized rats. To evoke a synchronous sensory input, the sciatic nerve was stimulated electrically and the resulting evoked spinal cord potential (SCP) recorded from the dorsal columns of the ipsilateral side. The waveshape of the SCP showed three components: an early positive peak (P1), representing the responses of the most rapidly conducting fibers, followed by two negative peaks (N1 and N2), which are mainly due to synaptic effects of the volley on dorsal horn cells located in dorsal grey matter. Laser irradiation at 50 mJ/pulse and above resulted in severe reduction in the amplitudes of N1 and N2. In contrast, there was either no reduction at all or only a slight decrease in the amplitude of P1. The selective loss of the synaptic field might be mediated by impairment of synaptic transmission or by loss of high threshold fiber input to dorsal horn neurones. In either event it is likely that the mechanism of the differential effects of laser irradiation on the components of the electrically evoked SCP is at least in part photothermally mediated, since intracord temperatures during laser application greatly exceeded the physiological range.     

Routing of transported materials in the dorsal root and nerve fiber branches of the dorsal root ganglion.

After injection of the L7 dorsal root ganglion with 3H-leucine, fast axoplasmic transport carries some 3--5 x more labeled materials down the sensory fibers branches entering the sciatic nerve as compared to the dorsal root fiber branches of the neurons. Freeze-substitution preparations taken from the two sides of the lumbar seventh dorsal root ganglia of cats and monkeys showed little difference in the histograms of nerve fiber diameters of the sensory nerve fiber branch of these neurons as compared to the dorsal root fiber branches. A similar density of microtubules and of neurofilaments in the dorsal root and sensory nerve fiber branches over a wide range of fiber diameters was found in electron micrograph preparations. In the absence of an anatomical difference in the fibers to account for the asymmetrical outflow, a functional explanation based on the transport filament model was advanced.     

Response of single alpha motoneurons to high-frequency pulse trains. Firing behavior and conduction block phenomenon

Studies were conducted on 25 cats to document the discharge rates of alpha motoneurons during stimulation of the sciatic nerve at frequencies from 100 to 10,000 pulses per second (pps). In addition, the feasibility of using high-frequency pulse trains to block the conduction of action potentials was investigated. Two cuff electrodes were placed around the proximal portion of the left sciatic nerve, and recordings of antidromic potentials were taken from single fibers of the L7 ventral root. When stimulating through the more proximal electrode, discharge rates were generally equal to or were subharmonics of the stimulation rate up to 1,000 pps. Firing often decreased in rate during 3-min runs. At 2,000-10,000 pps, fibers responded briefly at rates of several hundred per second but stopped firing within seconds after stimulus initiation. After cessation of response to the high-frequency pulse train, action potentials generated at 50 pps at the more distal electrode did not propagate to the recording electrodes. The 'electrical block' so induced was maintained for up to 20 min, and recovery following termination of the pulse train was complete within 1 s.     

Routing of transported materials in the dorsal root and nerve fiber branches of the dorsal root ganglion

After injection of the L7 dorsal root ganglion with ³H-leucine, fast axoplasmic transport carries some 3–5 × more labeled materials down the sensory fibers branches entering the sciatic nerve as compared to the dorsal root fiber branches of the neurons. Freeze-substitution preparations taken from the two sides of the lumbar seventh dorsal root ganglia of cats and monkeys showed little difference in the histograms of nerve fiber diameters of the sensory nerve fiber branch of these neurons as compared to the dorsal root fiber branches. A similar density of microtubules and of neurofilaments in the dorsal root and sensory nerve fiber branches over a wide range of fiber diameters was found in electron micrograph preparations. In the absence of an anatomical difference in the fibers to account for the asymmetrical outflow, a functional explanation based on the transport filament model was advanced.     

Distribution of myelinated and nonmyelinated nerve fibers of a cat cutaneous nerve in the spinal roots

The distribution of myelinated and nonmyelinated nerve fibers of the saphenous nerve of cats in the ventral and dorsal roots of the spinal cord was investigated by methods improving the signal—noise ratio in records of evoked responses from the nerve. The fibers of this nerve enter the spinal cord through roots of segments L_4–6. Nerve fibers with conduction velocities of between 80 and 0.38 m/sec were distributed in the dorsal roots of these segments. Four groups of nerve fibers with conduction velocities of 80–60, 40–30, 12.0–3.0, and 1.1–0.51 m/sec, possibly afferent in nature, were found in the ventral roots. The conditions of origin and detection of low-amplitude potentials in the roots of the spinal cord and the probable functional role of the nerve fibers in the ventral roots are discussed.Research Institute of Applied Mathematics and Cybernetics, N. I. Lobachevskii State University, Gor'kii. Translated from Neirofiziologiya, Vol. 7, No. 6, pp. 647–654, November–December, 1975.     

The role of sensory input in motor neuron sprouting control

Primary sensory neurons project to motor neurons directly or through interneurons and affect their activity. In our previous paper we showed that intramuscular sprouting can be affected by changing the sensory synaptic input to motor neurons. In this work, motor axon sprouting within a peripheral nerve (extramuscular sprouting) was induced by nerve injury at such a distance from muscle so as not to allow nerve-muscle trophic interactions. Two different procedures were carried out: (1) sciatic nerve crush and (2) sciatic nerve crush with homosegmental ipsilateral L3-L5 dorsal rhizotomy. The number of regenerating motor axons innervating extensor digitorum longus muscle was determined by in vivo muscle tension recordings and an index of their individual conduction rate was obtained by in vitro intracellular recordings of excitatory postsynaptic end-plate potentials in muscle fibers. The main findings were: (1) there are more regenerated axons distally from the lesion than parent axons proximally to the lesion (sprouting at the lesion); (2) sprouting at the lesion was negatively affected by homosegmental ipsilateral dorsal rhizotomy; (3) the number of motor axons innervating extensor digitorum longus muscle extrafusal fibers counted proximally to the lesion increased following nerve injury and regeneration but this did not occur when sensory input was lost. A transient innervation of extrafusal fibers by &#110 motor neurons may explain the increase of motor axons counted proximally to the lesion.     

Protein Synthesis and Rapid Axonal Transport During Regrowth of Dorsal Root Axons

Damage to the sciatic nerve produces significant changes in the relative synthesis rates of some proteins in dorsal root ganglia and in the amounts of some fast axonally transported proteins in both the sciatic nerve and dorsal roots. We have now analyzed protein synthesis and axonal transport after cutting the other branch of dorsal root ganglia neurons, the dorsal roots. Two to three weeks after cutting the dorsal roots, [35S]methionine was used to label proteins in the dorsal root ganglia in vitro. Proteins synthesized in the dorsal root ganglia and transported along the sciatic nerve were analyzed on two-dimensional gels. All of the proteins previously observed to change after sciatic nerve damage were included in this study. No significant changes in proteins synthesized in dorsal root ganglia or rapidly transported along the sciatic nerve were detected. Axon regrowth from cut dorsal roots was observed by light and electron microscopy. Either the response to dorsal root damage is too small to be detected by our methods or changes in protein synthesis and fast axonal transport are not necessary for axon regrowth. When such changes do occur they may still aid in regrowth or be necessary for later stages in regeneration.     

The role of sensory input in motor neuron sprouting control

Primary sensory neurons project to motor neurons directly or through interneurons and affect their activity. In our previous paper we showed that intramuscular sprouting can be affected by changing the sensory synaptic input to motor neurons. In this work, motor axon sprouting within a peripheral nerve (extramuscular sprouting) was induced by nerve injury at such a distance from muscle so as not to allow nerve-muscle trophic interactions. Two different procedures were carried out: (1) sciatic nerve crush and (2) sciatic nerve crush with homosegmental ipsilateral L3-L5 dorsal rhizotomy. The number of regenerating motor axons innervating extensor digitorum longus muscle was determined by in vivo muscle tension recordings and an index of their individual conduction rate was obtained by in vitro intracellular recordings of excitatory postsynaptic end-plate potentials in muscle fibers. The main findings were: (1) there are more regenerated axons distally from the lesion than parent axons proximally to the lesion (sprouting at the lesion); (2) sprouting at the lesion was negatively affected by homosegmental ipsilateral dorsal rhizotomy; (3) the number of motor axons innervating extensor digitorum longus muscle extrafusal fibers counted proximally to the lesion increased following nerve injury and regeneration but this did not occur when sensory input was lost. A transient innervation of extrafusal fibers by gamma motor neurons may explain the increase of motor axons counted proximally to the lesion.     

Uptake of Endoneurial Lipoprotein into Schwann Cells and Sensory Neurons Is Mediated by Low Density Lipoprotein Receptors and Stimulated After Axonal Injury

During Wallerian degeneration of rat sciatic nerve, the expression of apolipoprotein E increases and apolipoprotein E-containing endoneurial lipoproteins accumulate in the distal nerve segment. In established primary cultures dissociated from dorsal root ganglia, Schwann cells and sensory neurons internalized rhodamine-labeled lipoproteins isolated from crushed rat sciatic nerve as well as low density lipoprotein (LDL) from human serum. The uptake of endoneurial lipoproteins could be inhibited by an excess of LDL or at low temperature (4 degrees C). After transection of nerve fibers in dorsal root ganglia explant cultures, the uptake of lipoproteins was markedly stimulated in Schwann cells that were in close proximity to degenerating neurites. A specific monoclonal antibody directed to the bovine LDL receptor (clone C7) was shown to cross-react with LDL receptor preparations of rat endoneurial cells. LDL receptor immunoreactivity was expressed by cell bodies and processes of cultured Schwann cells, sensory neurons, and fibroblasts from dorsal root ganglia. Incubation of Schwann cells and neurons with the LDL receptor antibody strongly inhibited the uptake of endoneurial lipoproteins. Our results provide direct evidence for the important role of the LDL receptor-mediated pathway to internalize endoneurial lipoproteins into Schwann cells and peripheral neurons required for reuse of cholesterol and other lipids in myelin and plasma membrane biogenesis during nerve repair.     

Abnormal impulse discharge in primary afferent axons injured in the peripheral versus the central nervous system

Chronic injury to sensory axons in the rat peripheral nerve induces pathophysiologic changes in the axolemma at the cut nerve end, which are reflected in spontaneous ectopic impulse discharge and hyperexcitability to a range of depolarizing stimuli. We asked whether sensory axons injured in the central nervous system (CNS) also respond in this way. Primary afferent axons were severed in the sciatic nerve and, alternatively, in the midcervical or upper lumbar dorsal column (DC). Measurements of abnormal discharge from myelinated afferents showed high levels of spontaneous activity generated at the nerve injury site, especially during the period 3-16 days postoperatively, but comparatively little activity generated at the DC lesion site at any postoperative time. There was a corresponding difference in ectopic hyperexcitability to mechanical and adrenergic stimulation, and to depolarization with topical K+. DC lesion sites were not made more excitable by concurrent transection of the sciatic nerve, or by placing an autologous graft of excised sciatic nerve tissue into the DC defect at the time of initial surgery. Transection sites on dorsal roots L4 and L5 yielded abnormal discharge similar to that of sciatic nerve neuromas, indicating that the relative silence of DC transection sites was related to the CNS environment and not to position with respect to the sensory cell body.     

A technique for recording from intact phrenic nerve afferents

Recent evidence has suggested that phrenic nerve afferents can influence respiratory motor drive. This paper presents a technique whereby the activity of single phrenic nerve afferents can be recorded from uncut dorsal root filaments. Cervical dorsal roots 4, 5, and 6 were exposed by dorsal laminectomy in 10 anesthetized, spontaneously breathing cats. A stimulating electrode was placed on the right whole phrenic nerve low in the neck. The animal was then placed in a spinal suspension frame. Dissection of the dorsal root filaments was performed with probes made of fine tungsten wire. Single filaments were isolated intact from the dorsal root fascicles and placed across a tungsten electrode. Fiber classification was performed by determining conduction velocity. Monopolar recordings were made from a total of 38 fibers. Tonic activity was observed in 21, respiratory-related activity was evident in 15, and two fibers were silent but could be recruited by phrenic nerve stimulation. The conduction velocities ranged from 2.2 to 103 m/s. Approximately one-half of the fibers had conduction velocities of less than 20 m/s. This technique offers a way to record the activity of diaphragm afferents while maintaining the integrity of possible reflex pathways. Application of this method should prove helpful in elucidating the possible role of the various diaphragm afferents in the control of respiratory motor drive.     

Binding of γ-Aminobutyric AcidA Receptors to Tubulin

Abstract: The rate of axonal transport of tubulin, actin, and the neurofilament proteins was measured in the peripheral and central projections of the rat L5 dorsal root ganglion (DRG). [³⁵S]Methionine was injected into the DRG, and the "front" of the radiolabeled protein was located 7, 14, and 20 days postinjection. Transport rates calculated for the neurofilament triplet proteins, tubulin, and actin in the peripheral nerve were ∼ 1.5-fold faster than those in the dorsal root. A progressive decrease in the rate of transport was observed from 7 to 20 days after radiolabeling in both the central and peripheral directions (neurofilaments, ∼ 1.7-fold; tubulin/actin, 2.1-fold). A surgical preparation, leaving the peripheral sciatic nerve with predominantly sensory fibers, was the basis for ELISAs for phosphorylation-dependent immunoreactivity of the high-molecular-weight neurofilament protein. In both dorsal roots and peripheral sensory axons the degree of phosphorylation was greater in nerve segments further away from the cell bodies. The degree of phosphorylation-related immunoreactivity correlates with the slowing of transport of radiolabeled cytoskeletal protein.     

Patterns of slow transport in sensory nerves

An examination of the pattern of outflow of radioactivity in sciatic nerves was made at times from 1 to 82 days in the rat and up to 132 days in the cat after injecting the L5 and L7 dorsal root ganglia, respectively, with 3H-leucine. Slow waves moving at a rate of 1-2 mm/day were looked for on the basis of their reported presence in the motor fibers of the rat. A consistent pattern of slow waves was not seen in the cat or rat sensory fibers of the sciatic nerves nor was evidence of a slow wave found in the cat dorsal columns. Irregularities in the pattern of outflow which at times appeared as "waves" did so in an irregular fashion, a pattern inconsistent with a steady progression of slow waves in the fibers. The decrease of radioactivity appearing first near the ganglia helps create the impression of a wave along with irregular decreases in the overall levels of radio-activity with time. The results were explained on the basis of the unitary hypothesis. The labeled components are considered to be moved down the fiber by the fast transport mechanism, those components dropping off locally in the fibers early on, constituting the slow wave. As those components turn over locally in the various organelles of fiber and are further redistributed, they may at times give rise to what appears as waves.     

Ca2+ -free medium enhances the magnitude of slow peak in compound action potential of frog sciatic nerve in vitro

The present investigation was carried out to know the effect of Ca2+ on different peaks of compound action potential (CAP) representing the fibers having different conduction velocity. CAP was recorded from a thin bundle of nerve fibers obtained from desheathed frog sciatic nerve. Suction electrodes were used for stimulating and recording purposes. In Ca2+ -free amphibian Ringer, two distinct peaks (Peak-I and Peak-II) were observed. The threshold, conduction velocity (CV), amplitude and duration of Peak-I were 0.32 +/- 0.02 V, 56 +/- 3.0 m/sec, 2.1 +/- 0.2 mV and 0.75 +/- 0.1 ms, respectively. The Peak-II exhibited ten times greater threshold, eight times slower CV, three times lower amplitude and four times greater duration as compared to Peak-I. Addition of 2 mM Ca2+ in the bathing medium did not alter CAP parameters of Peak-I excepting 25% reduction in CV. But, in Peak-II there was 70-75% reduction in area and amplitude. The concentration-attenuation relation of Peak-II to various concentrations of Ca2+ was nonlinear and 50% depression occurred at 0.35 mM of Ca2+. Washing with Ca2+ -free solution with or without Mg2+ (2 mM)/verapamil (10 microM) could not reverse the Ca2+ -induced changes in Peak-II. Washing with Ca2+ -free solution containing EDTA restored 70% of the response. The results indicate that Ca2+ differentially influence fast and slow conducting fibers as the activity of slow conducting fibers is greatly suppressed by external calcium.     

RAPID AXOPLASMIC TRANSPORT OF PROTEINS IN REGENERATING SENSORY NERVE FIBERS

Abstract— Utilizing an in vitro labeling procedure, the proteins carried by rapid axoplasmic transport in normal and regenerating sensory fibers of the rat sciatic nerve were compared. No statistically significant differences were found when the total amount of transported protein was compared in control and sectioned nerves at times from 2 to 76 days following axotomy. Fractionation of labeled proteins on polyacrylamide slab gels enabled the identification of some 25 individual transported proteins. By this criterion, no differences were detectable in the composition of proteins synthesized in the dorsal root ganglia from which sectioned vs control sciatic nerves project. When the electrophoretic distributions of transported proteins from control and sectioned nerves were compared, significant' differences were observed. The appearance and disappearance of two proteins were temporally related to chromatolytic changes in the nerve cell body. In addition, the composition of transported proteins in undamaged control nerves contralateral to the sectioned nerves exhibited changes which were not observed in either normal control nerves or sectioned nerves. Changes in the composition of transported proteins as a function of time following the onset of chromatolysis may be involved in controlling nerve regeneration in sensory nerve fibers.     

Org.2766 improves functional and electrophysiological aspects of regenerating sciatic nerve in the rat

The beneficial effect of short-term (8 days) melanocortin therapy on regenerating peripheral nerves is demonstrated using functional and electrophysiological tests. Following a crush lesion of the rat sciatic nerve, recovery of sensory function is monitored by assessing the responsiveness of the rat to a small electric current applied to the footsole. Recovery of motor function is assessed by means of an analysis of walking patterns. Normalization of the walking pattern reflects reinnervation of different muscle groups. The motor and H-reflex related sensory nerve conduction velocity of the regenerated nerves are longitudinally investigated in the same rats in which the recovery of motor and sensory function had been assessed previously. Functional tests show an enhanced recovery under melanocortin therapy, but in the end both saline- and melanocortin-treated rats show 100% recovery. However, when compared to the contralateral sciatic nerve, in the peptide-treated animals motor nerve conduction in the regenerated nerves has fully recovered after about 90 days following the crush lesion and the sensory conduction after about 120 days, whereas in the saline-treated rats a deficit of 20-40% in both motor and sensory conduction remains. This difference is observed even 214 days following crush.     

Attenuation of phrenic motor discharge by phrenic nerve afferents

Short latency phrenic motor responses to phrenic nerve stimulation were studied in anesthetized, paralyzed cats. Electrical stimulation (0.2 ms, 0.01-10 mA, 2 Hz) of the right C5 phrenic rootlet during inspiration consistently elicited a transient reduction in the phrenic motor discharge. This attenuation occurred bilaterally with an onset latency of 8-12 ms and a duration of 8-30 ms. Section of the ipsilateral C4-C6 dorsal roots abolished the response to stimulation, thereby confirming the involvement of phrenic nerve afferent activity. Stimulation of the left C5 phrenic rootlet or the right thoracic phrenic nerve usually elicited similar inhibitory responses. The difference in onset latency of responses to cervical vs. thoracic phrenic nerve stimulation indicates activation of group III afferents with a peripheral conduction velocity of approximately 10 m/s. A much shorter latency response (5 ms) was evoked ipsilaterally by thoracic phrenic nerve stimulation. Section of either the C5 or C6 dorsal root altered the ipsilateral response so that it resembled the longer latency contralateral response. The low-stimulus threshold and short latency for the ipsilateral response to thoracic phrenic nerve stimulation suggest that it involves larger diameter fibers. Decerebration, decerebellation, and transection of the dorsal columns at C2 do not abolish the inhibitory phrenic-to-phrenic reflex.     

Qualitative analysis of proteins rapidly transported in ventral horn motoneurons and bidirectionally from dorsal root ganglia

Two-dimensional electrophoresis has allowed a higher-resolution comparison of rapid transport in ventral horn motoneurons and bidirectionally in dorsal root sensory neurons. Dorsal root ganglia 8 and 9, or hemisected spinal cords, from frog were selectively exposed in vitro to 35S-methionine. Transported, labelled proteins that accumulated in 3 mm segments proximal to ligatures on dorsal roots and spinal nerves or sciatic nerves were subjected to two-dimensional gel electrophoresis. Comparisons were made of fluorographic patterns from dried gels. Sixty-five species of proteins were found to be rapidly transported in both bifurcations of dorsal root sensory neurons. No abundant species of protein was rapidly transported in dorsal roots that was not also found in spinal nerves. A comparison of proteins rapidly transported in the sciatic nerve from ventral horn motoneurons with those from dorsal root sensory neurons yielded 50 common species of polypeptides. At most four minor species were possibly transported only in ventral horn motoneurons. An overall comparison indicates that at least 45 species of proteins, including all of the more abundantly transported ones, were consistently common to both dorsal root bifuractions and to ventral horn motoneurons. This appears to be the case despite the very different functions carried out by motoneurons and sensory neurons.     

Isolation and characterization of axolemma-enriched fractions from rabbit and bovine peripheral nerve

Axolemma-enriched fractions were isolated from bovine spinal accessory nerves, bovine intradural dorsal roots, and rabbit sciatic nerve by differential centrifugation and separation on a linear 10–40% sucrose (w/w) gradient. The fractions were enriched 4 to 10 fold in acetylcholinesterase, a biochemical marker for axolemma. Axolemma-enriched fractions isolated from uniformly well-myelinated fibers (bovine spinal accessory nerve) contained lower CNPase activity and higher acetylcholinesterase activity than comparable fractions isolated from variably myelinated fibers (rabbit sciatic nerve and bovine intradural roots). Separation by polyacrylamide electrophoresis showed that the molecular weight distribution of all peripheral nerve axolemma-enriched fractions was similar and ranged from 20 to over 150 kilodaltons. All axolemma-enriched fractions appeared to contain a small but variable amount of myelin-specific proteins. Based on biochemical properties, peripheral nerves containing uniformly well-myelinated fibers yield an axolemma-enriched fraction which is least contaminated with myelin-related membranes.     

ACTH/MSH like peptides in the treatment of cisplatin neuropathy.

The neurological toxicity seen in patients treated with cisplatin in most cases concerns ototoxicity and peripheral neuropathy. Thus far, the pathogenesis of cisplatin neuropathy remains obscure. Yet the fact that cisplatin affects mainly the sensory peripheral nerve fibers points towards an involvement of the dorsal root ganglia. In a rat model of cisplatin neuropathy, following a cumulative dose of approx. 12 mg/kg cisplatin the sensory nerve conduction velocity began to slow as compared to age-matched controls. Peptides derived from ACTH and MSH are known to exert neurotrophic effects. In vivo they facilitate postlesion repair mechanisms in the peripheral nervous system by enhancing the early sprouting response of the damaged nerve. Surprisingly, chronic treatment with a synthetic ACTH4-9 analog not only prevented cisplatin neurotoxicity following a low or high dose regimen, but also counteracted already existing cisplatin-induced neurotoxicity. Stimulated by these findings a randomized, double blind, placebo-controlled study was performed to assess the efficacy of the peptide in the prevention of cisplatin neuropathy in women suffering from ovarian cancer. The threshold of vibration perception (VPT) was used as the principal measure of neurotoxicity. Following 6 cycles of chemotherapy the VPT had increased more than 8-fold in women receiving placebo as co-medication. Whereas the VPT in women receiving 1 mg/m2 body surface ACTH4-9 analog before and after each cisplatin cycle only increased less than 2-fold. No side effects of the peptide treatment were observed and the clinical response to the chemotherapy was similar in all treatment groups. Collectively these preclinical and clinical data suggest that treatment based on non-endocrine fragments of ACTH/MSH may be a therapeutic option in the treatment of cisplatin neuropathy.