Janez Sketelj

Sex-related difference in collateral sprouting of nociceptive axons after peripheral nerve injury in the rat.

Possible sex-related differences in the extent of collateral sprouting of noninjured nociceptive axons after peripheral nerve injury were examined. In the first experiment, peroneal, tibial, and saphenous nerves were transected and ligated in female and male rats. Eight weeks after nerve injury, skin pinch tests revealed that the nociceptive area of the noninjured sural nerve in the instep skin expanded faster in females; the final result was a 30% larger increase in females than in males. In the second experiment, the end-to-side nerve anastomosis was used as a model for axon sprouting. In addition to the previous procedure, the end of an excised peroneal nerve segment was sutured to the side of the intact sural nerve. Eight weeks later, collateral sprouting of nociceptive axons into the anastomosed peroneal nerve segment was assessed by the nerve pinch test and axon counting. There was no significant difference with respect to the percentages of male and female rats with a positive nerve pinch test. The number of myelinated axons in the anastomosed nerve segment was significantly larger in female (456 +/- 217) than in male (202 +/- 150) rats, but the numbers of unmyelinated axons were not significantly different. In normal sural nerves, the numbers of either all myelinated axons or thin myelinated axons did not significantly differ between the two sexes. Therefore, the more extensive collateral axon sprouting observed in female than in male rats is probably due to the higher sprouting capacity of thin myelinated sensory axons in females.

Interneuronal signalling is involved in induction of collateral sprouting of nociceptive axons

Collateral sprouting of cutaneous nociceptive axons into the adjacent denervated skin critically depends on the nerve growth factor, presumably originating from the degenerated neural pathways and denervated skin. We hypothesised that the degenerated neural pathways are necessary, but not sufficient, to induce collateral sprouting of nociceptive axons, and, in addition, that the interaction between the injured and non-injured neurones within a dorsal root ganglion can trigger sprouting of nociceptive axons also in the absence of the denervated skin. End-to-side nerve anastomosis, made in female Wistar rats by suturing the end of an excised peroneal nerve segment to the side of the intact sural nerve, was used as a model for sprouting which allowed us to study the putative induction mechanisms separately. If the nerves adjacent to the sural nerve were transected concomitantly with the coaptation of the end-to-side anastomosis, robust nociceptive axon sprouting into the anastomosed nerve segment was observed by the nerve pinch test and counting of myelinated axons. Collateral sprouting did not occur, however, either if the cells in the anastomosed nerve segment were killed by freezing and thawing, or if the adjacent nerves had not been injured. However, if the ipsilateral dorsal cutaneous nerves, having their neurones in the same dorsal root ganglia as the sural nerve, were transected, but no other nerves were injured, then the sural nerve axons sprouted in abundance through the anastomosis even in the absence of denervated skin around the sural nerve terminals. From these results we suggest that cells (probably proliferating Schwann cells) in the degenerated neural pathways are necessary but not sufficient to induce collateral sprouting of nociceptive axons, and that interactions between the injured and non-injured neurones within the dorsal root ganglion (i.e. direct or indirect interneuronal signalling) are important in this regard.

Partial fast-to-slow conversion of regenerating rat fast-twitch muscle by chronic low-frequency stimulation.

Chronic low-frequency stimulation (CLFS) of rat fast-twitch muscles induces sequential transitions in myosin heavy chain (MHC) expression from MHCIIb → MHCIId/x → MHCIIa. However, the ‘final’ step of the fast-to-slow transition, i.e., the upregulation of MHCI, has been observed only after extremely long stimulation periods. Assuming that fibre degeneration/regeneration might be involved in the upregulation of slow myosin, we investigated the effects of CLFS on extensor digitorum longus (EDL) muscles regenerating after bupivacaine-induced fibre necrosis. Normal, non-regenerating muscles responded to both 30- and 60-day CLFS with fast MHC isoform transitions (MHCIIb → MHCIId → MHCIIa) and only slight increases in MHCI. CLFS of regenerating EDL muscles caused similar transitions among the fast isoforms but, in addition, caused significant increases in MHCI (to ∼30% relative concentration). Stimulation periods of 30 and 60 days induced similar changes in the regenerating bupivacaine-treated muscles, indicating that the upregulation of slow myosin was restricted to regenerating fibres, but only during an early stage of regeneration. These results suggest that satellite cells and/or regenerating fast rat muscle fibres are capable of switching directly to a slow program under the influence of CLFS and, therefore, appear to be more malleable than adult fibres.

Extent of Nociceptive Dermatomes in Adult Rats Is Not Primarily Maintained by Axonal Competition

Nociceptive innervation territories of individual peripheral and spinal nerves in the skin of the rat hind paw were investigated. In addition, the hypothesis that competitive interactions among the axons from adjacent dorsal root ganglia (DRG) play an important role in maintenance of dermatomal extent in adult animals was tested. The area of innervation territories of individual spinal and peripheral nerves was determined by nociceptive pinch test of the skin after extirpation of adjacent DRGs or transection of adjacent peripheral nerves, respectively. Positions of nociceptive dermatomes and innervation territories of peripheral nerves were similar to the territories innervated by the C-fibers described earlier by dye extravasation technique. In contrast, our results convincingly demonstrated substantial overlap of nociceptive (probably A delta) fibers from adjacent dermatomes in which the autonomous innervation areas were only about one-half of the maximal areas. Nociceptive territories of peripheral nerves overlapped, too. Accordingly, we could find no autonomous innervation area of the sural nerve. Two weeks after extirpation of adjacent DRGs, the area of each of the isolated dermatomes L3, L4, and L5 increased only by about 10%, and it did not change detectably during the next 6 months. The results of our study (a) support the view that innervation fields supplied by the nociceptive (probably A delta) fibers are greater and display more overlap than those supplied by the C-fibers of the same nerve and (b) suggest that axonal competition for innervation territory is not decisive for maintenance of dermatomal borders in the adult rat.

Chapter 26: Age-related differences in the reinnervation after peripheral nerve injury.

Numerous and extensive functional, structural, and biochemical changes characterize intact aged peripheral nervous system. Functional recovery after peripheral nerve injury depends on survival of injured neurons and functional reinnervation of target tissue by regeneration of injured axons and collateral sprouting of uninjured (intact) adjacent axons. The rate of axonal regeneration becomes slower and its extent (density and number of regenerating axons) decreases in aged animals. Aging also impairs terminal sprouting of regenerated axons and collateral sprouting of intact adjacent axons, thus further limiting target reinnervation and its functional recovery. Decreased survival of aged noninjured and injured neurons, limited intrinsic growth potential of neuron, alteration in its responsiveness to stimulatory or inhibitory environmental factors, and changes in the peripheral neural pathways and target tissues are possible reasons for impaired reinnervation after peripheral nerve injury in old age. The review of present data suggests that this impairment is mostly due to the age-related changes in the peripheral neural pathways and target tissues, and not due to the limited intrinsic growth capacity of neurons or their reduced responsiveness to trophic factors. Age-related alterations in the soluble target derived neurotrophic factors, like nerve growth factor, and nonsoluble extracellular matrix components of neural pathways, like laminin, might be important in this respect.

Fibre types and Myosin heavy chain expression in the Ocular Medial Rectus Muscle of the Adult Rat

Myosin heavy chain (MHC) expression was determined immunohistochemically in individual muscle fibre types characterised by activities of ATPase and the key oxidative and glycolytic enzymes in rat ocular medial rectus (MR) muscles. In the global layer (GL), glycolytic activity of muscle fibres was higher and oxidative activity lower, than in the orbital layer (OL). Muscle fibres in the former displayed rosette-like organisation with a slow fibre surrounded by several fast fibres, which expressed either MHCIIa or MHCIIb, but many co-expressed both isoforms. In the OL some slow fibres co-expressed MHCIIa. Extraocular MHC isoform (MHCeom) could not be determined immunohistochemically and no pure MHCIIx/d containing fibres were found, suggesting that these isoforms, demonstrated electrophoretically, are co-expressed with others. Slow muscle fibres in both layers co-expressed MHCβ slow, MHCα cardiac and MHC-slow tonic. Neonatal isoform (MHCneo) was co-expressed in several fast and slow muscle fibres in the orbital, but not global layer. Slow fibres in the GL displayed very low oxidative activity. Electrophoretic analysis of ocular MR muscle homogenates revealed that about 50% of total MHC was MHCIIb, MHCeom was quite prominent (25%), and MHCIIa, MHCIIx/d and MHCI contributed each about 8%. MHCneo, MHCslow tonic and MHCα cardiac could not be identified as separate bands.

Specific impulse patterns regulate acetylcholinesterase activity in skeletal muscles of rats and rabbits.

In rats, acetylcholinesterase (AChE) activity in the fast muscles is several times higher than in the slow soleus muscle. The hypothesis that specific neural impulse patterns in fast or slow muscles are responsible for different AChE activities was tested by altering the neural activation pattern in the fast extensor digitorum longus (EDL) muscle by chronic low‐frequency stimulation of its nerve. In addition, the soleus muscle was examined after hind limb immobilization, which changed its neural activation pattern from tonic to phasic. Myosin heavy‐chain (MHC) isoforms were analyzed by gel electrophoresis. Activity of the molecular forms of AChE was determined by velocity sedimentation. Low‐frequency stimulation of the rat EDL for 35 days shifted the profile of MHC II isoforms toward a slower MHCIIa isoform. Activity of the globular G1 and G4 molecular forms of AChE decreased by a factor of 4 and 10, respectively, and became comparable with those in the soleus muscle. After hind limb immobilization, the fast MHCIId isoform, which is not normally present, appeared in the soleus muscle. Activity of the globular G1 form of AChE increased approximately three times and approached the levels in the fast EDL muscle. In the rabbit, on the contrary to the rat, activity of the globular forms of AChE in a fast muscle increased after low‐frequency stimulation. The results demonstrate that specific neural activation patterns regulate AChE activity in muscles. Great differences, however, exist among different mammalian species in regard to muscle AChE regulation. J. Neurosci. Res. 47:49–57, 1997.

Collateral sprouting of sensory axons after end-to-side nerve coaptation—A longitudinal study in the rat

The end-to-side nerve coaptation is able to induce collateral sprouting of axons from the donor nerve and to provide functional reinnervation of the target tissue. Sensory axon sprouting and its effects on the donor nerve up to 9 months after the end-to-side nerve coaptation were studied in the rat. Peroneal, tibial and saphenous nerves were transected and ligated, and the distal stump of the transected peroneal nerve was sutured to the side of the uninjured sural nerve. The average skin area of the residual sensitivity to pinch due to the axons sprouting through the recipient peroneal nerve did not change statistically significantly between 4 and 9 months after surgery. Axon counting, measurements of compound action potentials and retrograde neuron labeling indicate that the sprouting of the myelinated sensory axons and unmyelinated axons through the recipient nerve was largely completed by 2 months and 4 months after the end-to-side nerve coaptation, respectively, and remained stable thereafter for at least 9 months. A decrease in the amplitude and area of the CAP of myelinated fibers, observed in the donor nerve up to 4 months after surgery, was probably due to mild degeneration of nerve fibers and a tendency of the diameter of myelinated axons to decline. However, no significant changes in functional, electrophysiological or morphological properties of the donor nerve could be observed at the end of the observational period, indicating that end-to-side nerve coaptation has no detrimental effect on the donor nerve on a long-term scale.

The effect of hyperbaric oxygen treatment on early regeneration of sensory axons after nerve crush in the rat

Abstract  The effect of hyperbaric oxygen treatment (HBO) on sensory axon regeneration was examined in the rat. The sciatic nerve was crushed in both legs. In addition, the distal stump of the sural nerve on one side was made acellular and its blood perfusion was compromised by freezing and thawing. Two experimental groups received hyperbaric exposures (2.5 ATA) to either compressed air (pO2 = 0.5 ATA) or 100% oxygen (pO2 = 2.5 ATA) 90 minutes per day for 6 days. Sensory axon regeneration in the sural nerve was thereafter assessed by the nerve pinch test and immunohistochemical reaction to neurofilament. HBO treatment increased the distances reached by the fastest regenerating sensory axons by about 15% in the distal nerve segments with preserved and with compromised blood perfusion. There was no significant difference between the rats treated with different oxygen tensions. The total number of regenerated axons in the distal sural nerve segments after a simple crush injury was not affected, whereas in the nerve segments with compromised blood perfusion treated by the higher pO2, the axon number was about 30% lower than that in the control group. It is concluded that the beneficial effect of HBO on sensory axon regeneration is not dose‐dependent between 0.5 and 2.5 ATA pO2. Although the exposure to 2.5 ATA of pO2 moderately enhanced early regeneration of the fastest sensory axons, it decreased the number of regenerating axons in the injured nerves with compromised blood perfusion of the distal nerve stump.

Adaptive range of myosin heavy chain expression in regenerating soleus is broader than in mature muscle

SummaryIn adult rat muscles experimentally exposed to various patterns of activation, expression of myosin heavy chain isoforms changes, but only within a certain adaptive range. It is characteristic and different in fast or slow muscles. This may be due either to different intrinsic properties of the myogenic cells of the two types of muscles or to extrinsic factors. To test these assumptions, either rat soleus or extensor digitorum longus muscles were injured and transplanted to the bed of the extensor digitorum longus muscle. They regenerated and were reinnervated by the extensor digitorum longus nerve. Expression of myosin heavy chain isoforms was demonstrated immunohistochemically and by in situ hybridization, and analysed by SDS-gel electrophoresis. Three months after cross-transplantation, regenerated soleus expressed all adult myosin heavy chain isoforms, including the myosin heavy chain-2B. The latter was detected in about 50% of muscle fibres and contributed about 10–20% of all myosin heavy chains. The same percentage of myosin heavy chain-2B was found in regenerated extensor digitorum longus. In this regard therefore, the adaptive range of the regenerated soleus muscle was not significantly different from that of the extensor digitorum longus regenerating under the same conditions. This indicates that restriction of the adaptive range in a mature soleus muscle is not due to intrinsic properties of its myogenic cells. It is probably imposed by an extrinsic factor leading to irreversible shut-down of individual myosin heavy chain genes. On the other hand, myosin heavy chain-1 expression was significantly greater in the regenerated soleus than in the extensor digitorum longus innervated by the same nerve. Myosin heavy chain-1 and myosin heavy chain-2B were co-expressed in some regenerated soleus muscle fibres.