Mechano- and thermosensitivity of injured muscle afferents 20 to 80 days after nerve injury.

Chronic injury of limb nerves leading to neuropathic pain affects deep somatic nerves. Here the functional properties of injured afferent fibers in the lateral gastrocnemius-soleus nerve were investigated 20 and 80 days after suturing the central stump of this muscle nerve to the distal stump of the sural nerve in anesthetized rats. Neurophysiological recordings were made from afferent axons identified in either the sciatic nerve (87 A-, 63 C-fibers) or the dorsal root L4/L5 (52 A-, 26 C-fibers) by electrical stimulation of the injured nerve. About 70% of the functionally identified A-fibers had regenerated into skin by 80 days after nerve suture; the remaining A-fibers could be activated only from the injured nerve. In contrast, 93% of the functionally identified C-fibers could only be activated from the injured sural nerve after 80 days. Nearly half of the injured A- (45%) and C-fibers (44%) exhibited ongoing and/or mechanically or thermally evoked activity. Because ~50% of the A- and C-fibers are somatomotor or sympathetic postganglionic axons, respectively, probably all injured muscle afferent A- and C-fibers developed ectopic activity. Ongoing activity was present in 17% of the A- and 46% of the C-fibers. Mechanosensitivity was present in most injured A- (99%) and C-fibers (85%), whereas thermosensitivity was more common in C-fibers (cold 46%, heat 47%) than in A-fibers (cold 18%, heat 12%). Practically all thermosensitive A-fibers and C-fibers were also mechanosensitive. Thus, unlike cutaneous axons, almost all A- and C-fibers afferents in injured muscle nerves demonstrate ectopic activity, even chronically after nerve injury. NEW & NOTEWORTHY After chronic injury of a muscle nerve, allowing the nerve fibers to regenerate to the target tissue, 1) most afferent A-fibers are mechanosensitive and regenerate to the target tissue; 2) ectopic ongoing activity, cold sensitivity, and heat sensitivity significantly decrease with time after injury in A-afferents; 3) most afferent C-fibers do not regenerate to the target tissue; and 4) injured C-afferents maintain the patterns of ectopic discharge properties they already show soon after nerve injury.

Responses of intact and injured sural nerve fibers to cooling and menthol.

Intact and injured cutaneous C-fibers in the rat sural nerve are cold sensitive, heat sensitive, and/or mechanosensitive. Cold-sensitive fibers are either low-threshold type 1 cold sensitive or high-threshold type 2 cold sensitive. The hypothesis was tested, in intact and injured afferent nerve fibers, that low-threshold cold-sensitive afferent nerve fibers are activated by the transient receptor potential melastatin 8 (TRPM8) agonist menthol, whereas high-threshold cold-sensitive C-fibers and cold-insensitive afferent nerve fibers are menthol insensitive. In anesthetized rats, activity was recorded from afferent nerve fibers in strands isolated from the sural nerve, which was either intact or crushed 6-12 days before the experiment distal to the recording site. In all, 77 functionally identified afferent C-fibers (30 intact fibers, 47 injured fibers) and 34 functionally characterized A-fibers (11 intact fibers, 23 injured fibers) were tested for their responses to menthol applied to their receptive fields either in the skin (10 or 20%) or in the nerve (4 or 8 mM). Menthol activated all intact (n = 12) and 90% of injured (n = 20/22) type 1 cold-sensitive C-fibers; it activated no intact type 2 cold-sensitive C-fibers (n = 7) and 1/11 injured type 2 cold-sensitive C-fibers. Neither intact nor injured heat- and/or mechanosensitive cold-insensitive C-fibers (n = 25) and almost no A-fibers (n = 2/34) were activated by menthol. These results strongly argue that cutaneous type 1 cold-sensitive afferent fibers are nonnociceptive cold fibers that use the TRPM8 transduction channel.

Reflex inhibition of cutaneous and muscle vasoconstrictor neurons during stimulation of cutaneous and muscle nociceptors.

Cutaneous (CVC) and muscle (MVC) vasoconstrictor neurons exhibit typical reflex patterns to physiological stimulation of somatic and visceral afferent neurons. Here we tested the hypothesis that CVC neurons are inhibited by stimulation of cutaneous nociceptors but not of muscle nociceptors and that MVC neurons are inhibited by stimulation of muscle nociceptors but not of cutaneous nociceptors. Activity in the vasoconstrictor neurons was recorded from postganglionic axons isolated from the sural nerve or the lateral gastrocnemius-soleus nerve in anesthetized rats. The nociceptive afferents were excited by mechanical stimulation of the toes of the ipsilateral hindpaw (skin), by hypertonic saline injected into the ipsi- or contralateral gastrocnemius-soleus muscle, or by heat or noxious cold stimuli applied to the axons in the common peroneal nerve or tibial nerve. The results show that CVC neurons are inhibited by noxious stimulation of skin but not by noxious stimulation of skeletal muscle and that MVC neurons are inhibited by noxious stimulation of skeletal muscle but not by noxious stimulation of skin. These inhibitory reflexes are mostly lateralized and are most likely organized in the spinal cord. Stimulation of nociceptive cold-sensitive afferents does not elicit inhibitory or excitatory reflexes in CVC or MVC neurons. The reflex inhibition of activity in CVC or MVC neurons generated by stimulation of nociceptive cutaneous or muscle afferents during tissue injury leads to local increase of blood flow, resulting in an increase of transport of immunocompetent cells, proteins, and oxygen to the site of injury and enhancing the processes of healing.

Mechano- and thermosensitivity of injured muscle afferents.

Injury of limb nerves leading to neuropathic pain mostly affects deep somatic nerves including muscle nerves. Here, we investigated the functional properties of injured afferent fibers innervating the lateral gastrocnemius-soleus muscle 4-13 h [time period (TP) I] and 4-7 days (TP II) after nerve crush in anesthetized rats using neurophysiological recordings from either the sciatic nerve (165 A-, 137 C-fibers) or the dorsal root L(5) (43 A-, 28 C-fibers). Ongoing activity and responses to mechanical or thermal stimulation of the injury site of the nerve were studied quantitatively. Of the electrically identified A- and C-fibers, 5 and 38% exhibited ectopic activity, respectively, in TP I and 51 and 61%, respectively, in TP II. Thus all afferent fibers in an injured muscle nerve developed ectopic activity since ∼ 50% of the fibers in a muscle nerve are somatomotor or sympathetic postganglionic. Ongoing activity was present in 50% of the afferent A-fibers (TP II) and in 53-56% of the afferent C-fibers (TP I and II). In TP II, mechanical, cold, and heat sensitivity were present in 91, 63, and 52% of the afferent A-fibers and in 50, 40, and 66% of the afferent C-fibers. The cold and heat activation thresholds were 5-27 and 35-48°C, respectively, covering the noxious and innocuous range. Most afferent fibers showed combinations of these sensitivities. Mechano- and cold sensitivity had a significantly higher representation in A- than in C-fibers, but heat sensitivity had a significantly higher representation in C- than in A-fibers. These functional differences between A- and C-fibers applied to large- as well as small-diameter A-fibers. Comparing the functional properties of injured muscle A- and C-afferents with those of injured cutaneous A- and C-afferents shows that both populations of injured afferent neurons behave differently in several aspects.

Axonal thermosensitivity and mechanosensitivity of cutaneous afferent neurons.

We hypothesized that cutaneous afferent myelinated fibers (A-fibers) and afferent unmyelinated fibers (C-fibers) respond to the same natural stimuli applied to their axons as to their terminals in the skin. In anesthetized rats, activity was recorded from afferent axons in strands isolated proximally from the sural nerve. Mechanical, cold or heat stimuli were applied to the skin or along a 15-mm length of the distal sural nerve. One-hundred and eighteen A-fibers and 109 C-fibers were characterized by their conduction velocity and/or shape of their action potentials, and by their responses to natural stimulation of the skin. Then, these fibers were tested for their responses to the same stimuli applied to the nerve. In some cases, the nerve was crushed distally after the nerve fibers had been characterized by their responses to physiological stimulation of the skin, and the responses to stimuli applied to the nerve proximal to the lesion were tested again. Almost all non-nociceptive cold-sensitive (type 1) C-fibers (97%) could be activated by cold stimuli applied to the nerve. Of nociceptive cold-sensitive (type 2) C-fibers, 39% were activated by cold stimuli applied to the nerve. Furthermore, 34% of heat-sensitive C-fibers could be activated by heating the nerve. In contrast, only 2-4% of mechanosensitive A-fibers and C-fibers responded to mechanical stimuli applied to the nerve. In conclusion, cold and heat sensitivity of cutaneous afferent neurons is not restricted to their terminals in the skin, but often extends along the axons in the nerve. Mechanosensitivity is restricted to the afferent endings in the skin.

Ganglionic transmission in a vasomotor pathway studied in vivo.

Intracellular recordings were made in vivo from 40 spontaneously active cells in the third lumbar sympathetic ganglion of urethane-anaesthetized rats. In 38/40 cells ongoing action potentials showed strong cardiac rhythmicity (93.4 +/- 1.9% modulation) indicating high barosensitivity and probable muscle vasoconstrictor (MVC) function. Subthreshold excitatory postsynaptic potentials (EPSPs) showed the same pattern. The 38 barosensitive neurons fired action potentials at 2.9 +/- 0.3 Hz. All action potentials were triggered by EPSPs, most of which were unitary events. Calculations indicated that <5% of action potentials were triggered by summation of otherwise subthreshold EPSPs. 'Dominant' synaptic inputs with a high safety factor were identified, confirming previous work. These were active in 24/38 cells and accounted for 32% of all action potentials; other ('secondary') inputs drove the remainder. Inputs (21 dominant, 19 secondary) attributed to single preganglionic neurons fired at 1.38 +/- 0.16 Hz. An average of two to three preganglionic neurons were estimated to drive each ganglion cell's action potentials. When cells were held hyperpolarized to block spiking, a range of spontaneous EPSP amplitudes was revealed. Threshold equivalent was defined as the membrane potential value that was exceeded by spontaneous EPSPs at the same frequency as the cell's original firing rate. In 10/12 cells examined, a continuum of EPSP amplitudes overlapped threshold equivalent. Small changes in cell excitability could therefore raise or lower the percentage of preganglionic inputs triggering action potentials. The results indicate that vasoconstrictor ganglion cells in vivo mostly behave not as 1:1 relays, but as continuously variable gates.

Functional properties of cutaneous A- and C-fibers 1-15 months after a nerve lesion.

The functional properties of cutaneous afferent fibers were investigated 1-15 mo after nerve lesions, which allowed regeneration into denervated skin. After crushing or transection and resuturing the rat sural nerve, ongoing activity and responses to cold, heat, and mechanical stimuli presented to the denervated skin or to the nerve distal to the lesion were examined in 273 A-fibers and 211 C-fibers. Reinnervation of skin by A-fibers was largely complete by 1-4 mo after crushing but incomplete after transection and resuturing. A few A-fibers could be activated from the nerve trunk, even after 10-15 mo. Almost all regenerated A-fibers were mechanosensitive and about 6% were cold- or heat-sensitive. A few A-fibers had ongoing activity after nerve crush. Only 15-35% of C-fibers could be activated at 1-4 mo, but 60% were excited from the skin at 10-15 mo, when many also had receptive fields within the lesioned nerve. The remaining C-fibers had receptive fields only within the nerve trunk. Responses of both intraneural and intradermal endings of C-fibers could be classified into functional groups similar to those of C-fibers in control nerves to cutaneous stimuli. The frequency of afferent C-fibers with ongoing activity that were not highly cold sensitive was 45%. We conclude that the functional characteristics of afferent A- and C-fibers are expressed by regenerating nerve endings, even when they do not reinnervate their target tissue. The reinnervation of skin by afferent C-fibers is extremely slow and may never recover to normal.

Mechano- and thermosensitivity of regenerating cutaneous afferent nerve fibers.

Crush lesion of a skin nerve is followed by sprouting of myelinated (A) and unmyelinated (C) afferent fibers into the distal nerve stump. Here, we investigate quantitatively both ongoing activity and activity evoked by mechanical or thermal stimulation of the nerve in 43 A- and 135 C-fibers after crush lesion of the sural nerve using neurophysiological recordings in anesthetized rats. The discharge patterns in the injured afferent nerve fibers and in intact (control) afferent nerve fibers were compared. (1) Almost all (98%) A-fibers were mechanosensitive, some of them exhibited additionally weak cold/heat sensitivity; 7% had ongoing activity. (2) Three patterns of physiologically evoked activity were present in the lesioned C-fibers: (a) C-fibers with type 1 cold sensitivity (low cold threshold, inhibition on heating, high level of ongoing and cold-evoked activity; 23%): almost all of them were mechanoinsensitive and 40% of them were additionally heat-sensitive; (b) C-fibers with type 2 cold sensitivity (high cold threshold, low level of ongoing and cold-evoked activity; 23%). All of them were excited by mechanical and/or heat stimuli; (c) cold-insensitive C-fibers (54%), which were heat- and/or mechanosensitive. (3) The proportions of C-fibers exhibiting these three patterns of discharge to physiological stimuli were almost identical in the population of injured C-fibers and in a population of 91 intact cutaneous C-fibers. 4. Ongoing activity was present in 56% of the lesioned C-fibers. Incidence and rate of ongoing activity were the same in the populations of lesioned and intact type 1 cold-sensitive C-fibers. The incidence (but not rate) of ongoing activity was significantly higher in lesioned type 2 cold-sensitive and cold insensitive C-fibers than in the corresponding populations of intact C-fibers (42/93 fibers vs. 11/72 fibers).

Peripheral thermoreceptors in innocuous temperature detection.

The mammalian skin is innervated by cold-sensitive afferent neurons. These neurons exhibit ongoing activity at temperatures between ~10 and 42°C, are activated by innocuous cold stimuli, inhibited by warm stimuli and are mechanoinsensitive. Their axons are small-diameter myelinated (Aδ-) fibers in primates and unmyelinated (C-) fibers in nonprimate mammals. The mammalian skin is innervated by warm-sensitive afferent neurons. The density of innervation by these neurons is lower than that by cold-sensitive afferents. They exhibit ongoing activity between ~38 and 48°C, are activated by warm stimuli, inhibited by cold stimuli, and are mechanoinsensitive. Their axons are unmyelinated (C-) fibers. Cold-sensitive unmyelinated afferent neurons exhibit prominent cold sensitivity of their axons (in rats). The discharge pattern of the cutaneous cold-sensitive afferent neurons is fully preserved after nerve injury. Ongoing impulse activity and cold-evoked impulses originate ectopically at the nerve injury site. Deep somatic tissues and viscera are innervated by thermosensitive afferent neurons. Most are warm-sensitive and mechanoinsensitive and have unmyelinated axons. These afferent neurons have only rarely and incompletely been studied, e.g., in the upper gastrointestinal tract, the liver (both vagal afferents), the dorsal abdominal wall, and the skeletal muscle. Spinal cord warm sensitivity may be mediated by cutaneous afferent neurons with unmyelinated axons that are excited by spinal cord warming.

Behavioral and sensory changes after direct ischemia-reperfusion injury in rats.

Complex regional pain syndromes (CRPS) are disabling pain syndromes that can develop after trauma or minor tissue injury affecting a limb. Characteristics of CRPS are sensory signs and symptoms, autonomic abnormalities, trophic changes and an impaired motor function. Pathophysiological mechanisms for the development of CRPS are still a matter of investigation. Based on clinical data and investigations of CRPS patients it is hypothesized that tissue hypoxia and inflammation are important for the development of CRPS. The aim of the current study was therefore to examine if direct ischemia-reperfusion injury can induce behavior in rats with symptoms present in patients with CRPS. After baseline behavior measurements the femoral artery of Wistar rats was ligated for 3h with consecutive reperfusion. Sham-operated rats underwent the same preparation except ligation of the artery. Subsequent behavioral testing (observations of spontaneous pain behavior, paw withdrawal to mechanical, noxious mechanical, cold and heat stimuli) was performed up to two months after surgery. Both in rats that underwent ischemia and in sham-operated rats no obvious changes of hindpaw tissue were observed after ischemia-reperfusion injury (trophic changes, edema, differences in skin color or temperature). In behavioral tests only minor changes were observed, these being not different between postischemic rats and sham-operated rats. Using Wistar rats, our data do not support the idea that an ischemia-reperfusion injury can play a major role in the development of CRPS.

The Sacral Autonomic Outflow Is Spinal, but Not "Sympathetic".

A recent article published in a high-profile journal proposed to reclassify the sacral autonomic outflow as being part of the sympathetic system. In this commentary, arguments against this erroneous proposal are provided. Anat Rec, 300:1369-1370, 2017. © 2017 Wiley Periodicals, Inc.

Autonomic dysfunction in rheumatic diseases.

Patients who have rheumatic diseases often present with dysfunctions that are related to the autonomic nervous system (ANS) and are due to peripheral autonomic neuropathy or central changes. This article describes the prevalence of autonomic dysfunctions in patients who have rheumatic diseases. In the second part of this article, another form of ANS dysfunction-complex regional pain syndromes-is demonstrated. Clinically, these syndromes are characterized by pain (spontaneous, hyperalgesia, allodynia); active movement disorders, including an increased physiologic tremor, abnormal regulation of blood flow and sweating, edema of skin and subcutaneous tissues; and trophic changes of skin, appendages of skin, and subcutaneous tissues. In conclusion, this discussion shows that alterations of the ANS occur in rheumatic and related diseases, that these alterations may be involved in the pathogenesis of these diseases, and that we need more refined methods to study the changes that are related to the ANS.

Complex regional pain syndrome: mystery explained?

Complex regional pain syndrome (CRPS) is the result of changes to the somatosensory systems that process noxious, tactile, and thermal information; to the sympathetic systems that innervate skin (blood vessels, sweat glands); and to the somatomotor systems. The changes suggest that the CNS representations of the systems have been altered. Patients with CRPS also have peripheral changes (eg, oedema, signs of inflammation, sympathetic-afferent coupling [the basis for sympathetically maintained pain], and trophic changes) that cannot be explained by central changes. On the basis of clinical observation and research in human beings and animals, we hypothesise that CRPS is a systemic disease involving the CNS and peripheral nervous system. The most important question for future research is what causes CRPS? In this article, we suggest a change to the focus of research efforts and treatment. We also suggest there be diagnostic reclassification and redefinition of CRPS.

Receptive properties of pial afferents.

The blood vessels and the pial surface of the brain, spinal cord and its roots are innervated by primary afferent neurones. Here, we have electrophysiologically characterized the functional properties of a subpopulation of these afferent fibres that supply the ventral roots of the cat sacral spinal cord. We have taken advantage of the unique anatomical arrangement of these primary afferent neurones which have their central axon in the dorsal root and project with their peripheral process into the segmental ventral root. In 10 experiments, 14 units were recorded in the dorsal root S2 which responded to electrical stimulation of the segmental ventral root. As judged by their conduction velocity ranging from 0.1 to 2.3 m/sec, all fibres were unmyelinated. In 4 cases a spot-like receptive field was located on the root where the units were reproducibly activated by mechanical stimuli, the most effective being a slight stretch. In two units tested, topical application of hypertonic saline onto the receptive field, but not at other portions of the axon elicited a long-lasting vigorous discharge with intermittent bursts. There was no obvious association of the receptive field with small blood vessels. In 5 of the 14 units including 2 with a mechanosensitive receptive field we observed latency jumps of the action potential with electrical stimulation of the ventral root close to the dorsal root ganglion. In some of these units latency jumps were also observed at other positions when the stimulation electrodes were moved centrally towards the spinal cord. We conclude that a subpopulation of unmyelinated fibres in the spinal ventral root are primary afferents innervating the root proper or its sheath.(ABSTRACT TRUNCATED AT 250 WORDS)