Dynamics of the recovery of the structure and function of the sciatic nerve and skin receptors in reinnervation of the hindlimb of white rats

The dynamics of the structural and functional repair of nerve trunks and receptor endings in the rat foot skin was studied on the model of sutureless connection of the damaged nerve by and implanted arterial vessel. Neurohistological and neurophysiological methods were used to show that the anatomical repair of the nerve integrity, maturation of nerve fibers and formation of receptor endings preceded their functional restoration. Terms of myelinization of regenerating axons, their diameters and initial stages of the formation of receptors in the foot skin were established. The restoration of the skin receptor function was shown to occur within 11-13 months after operation.     

Restoration of the innervation of the extremity of the rat after joining the ends of the damaged nerve with a microsurgical suture

Morphofunctional restoration of the cut ischemic nerve has been studied after its connection by means of a microsurgical suture. The investigations have been performed on 20 male rats. In 6 months after the operation the average diameter of the regenerating nervous fibers is 5.0-6.5 mcm and, as in an intact nerve they have a wavy course. Myelin decay products and inflammatory infiltrates are revealed in the nerve but sometimes. All the parts of the plantar skin are well reinnervated. Cutaneous epidermis of the sole and the digital pads possesses an extremely rich innervation. Characteristics of M-responses, registered from the m. gastrocnemius, approaches the norm in 2 months after the operation. In 10% of fibers of the regenerating ischiatic nerve impulse activity begins to be registered in 20-30 days after the operation. Gradually the borders of the receptor fields become wider and in 3 months they spread all over the whole sole. In 5-6 months the impulse reaction of the regenerating nerve fibers does not differ from that in non-operated animals. Thus, use of the microsurgical suture results in a successful regeneration of the rat ischemic nerve and in a rather short time. The restorative dynamics of the extremity tissue innervation can be presented as following: at first somatic muscles get innervation and then, as the afferent fibers grow in the periphery, the plantar skin and the skin of the digital pads is the last to get innervation.     

Sensory restoration of the skin graft on a free muscle flap: experimental rabbit study.

Transplantation of a muscle flap with free skin graft for wound coverage is a common procedure in reconstructive microsurgery. However, the grafted skin has little or no sensation. Restoration of the sensibility of the grafted skin on the transferred muscle is critically important, especially in palmar hand, plantar foot, heel, and oral cavity reconstruction. The purpose of this study was to investigate the possibility of sensory restoration of the grafted skin on a trimmed muscle surface that has been sensory neurotized after sensory nerve-to-motor nerve transfer, using the rabbit gracilis muscle as an animal model. The ipsilateral saphenous nerve (sensory) was transferred to the motor nerve of the gracilis muscle for sensory neurotization. A 4 x 4-cm2 area of skin island over the midportion of the gracilis muscle was harvested as a full-thickness skin graft. The upper half of the gracilis muscle was then excised, becoming a rough surface. The harvested skin was reapplied on the trimmed rough surface of the muscle. After 6 months, retrograde and antegrade horseradish peroxidase labeling studies were performed through skin and muscle injection. The group with a free skin graft was compared with the group with an intact surface of the gracilis muscle. This study clearly shows that sensory nerves can regenerate and penetrate into the trimmed muscle surface and grow into the overlying grafted skin. However, if the muscle surface is intact as with the compared group, sensory reinnervation of the grafted skin is not possible.     

Time Course and Characteristics of the Capacity of Sensory Nerves to Reinnervate Skin Territories outside Their Normal Innervation Zones

Factors involved in the outcome of regeneration of the saphenous nerve after a cut or crush lesion were studied in adult rats with electrophysiological recordings of low-threshold mechanoreceptor activity and plasma extravasation of Evans blue after electrical nerve stimulation that activated C fibers.

In the first series of experiments, saphenous and sciatic nerve section was combined with anastomosis of the transected proximal end of the saphenous nerve to the distal end of the cut tibial nerve. Regeneration of saphenous nerve fibers involved in plasma extravasation and low-threshold mechanoreceptor activity in the glabrous skin was observed 13 weeks after nerve anastomosis. Substance P-, calcitonin gene-related peptide-, and protein gene product 9.5 (PGP-9.5)-immunoreactive (IR) thin epidermal and dermal nerve endings, as well as coarse dermal PGP-9.5-IR nerve fibers and Meissner corpuscles and Merkel cell-neurite-like complexes, were observed in the reinnervated glabrous skin at this time.

In a second series of experiments, the time course of the regeneration of saphenous nerve axons to the permanently sciatic-nerve-denervated foot sole was examined. Saphenous-nerve-induced plasma extravasation and low-threshold mechanoreceptor activity in the saphenous nerve were found in the normal saphenous nerve territory 2, 3, 4, and 6 weeks after sciatic nerve cut combined with saphenous nerve crush in the left hindlimb. Saphenous-nerve-induced plasma extravasation was also present in the glabrous skin normally innervated by the sciatic nerve 3, 4, and 6 weeks after the sciatic cut/saphenous crush lesion. However, no low-threshold mechanoreceptor activity was detected in the saphenous nerve when the glabrous skin area was stimulated.

In a third series of experiments, the fate of the expansion of the saphenous nerve territory after saphenous nerve crush was examined when the crushed sciatic nerve had been allowed to regenerate. Nerve fibers involved in plasma extravasation were observed in the glabrous skin of the hindpaw after saphenous nerve, as well as after tibial nerve, C-fiber stimulation 3, 12, and 43 weeks after the saphenous crush/sciatic crush lesion.

Low-threshold mechanoreceptors from the regenerated saphenous nerve, which primarily innervates hairy skin, seem to be functional in the glabrous skin if the axons are guided by the transected tibial nerve by anastomosis. Furthermore, the results indicate that fibers from the regenerating saphenous nerve that have extended into denervated glabrous skin areas can exist even if sciatic nerve axons are allowed to grow back to their original territory.     

Anti-CD40 ligand antibody permits regeneration through peripheral nerve allografts in a nonhuman primate model

Systemic immunosuppression is typically required to prevent allograft rejection. Antibody-based therapies that induce immune unresponsiveness represent an appealing alternative to nonspecific immunosuppression, which is often associated with significant morbidity. In mice, successful prevention of nerve allograft rejection has been demonstrated through interference with the CD40/CD40 ligand interaction. This study investigated the effectiveness of anti-CD40 ligand monoclonal antibody as single-agent therapy in preventing rejection and supporting nerve regeneration across long nerve allografts in nonhuman primates. Twelve outbred cynomolgus macaques were arranged into six genetically mismatched pairs, with each animal receiving a 5-cm ulnar nerve allograft in the right arm and a 5-cm autograft in the left arm. Mixed lymphocyte reaction assays were used to assess resulting immune unresponsiveness. Treated animals (n = 10) received anti-CD40 ligand monoclonal antibody 10 mg/kg one time, locally applied, and 20 mg/kg systemically on postoperative days 0, 1, 3, 10, 18, and 28, and then monthly. Untreated animals (n = 2) served as the untreated controls. At 4 or 6 months after transplantation, nerves were harvested for histological analysis. Four treated animals underwent an additional challenge after cessation of anti-CD40 ligand monoclonal antibody therapy and nerve graft harvests. Autogenous and allogeneic skin and nerve inlay grafting was performed to assess the permanence of immune unresponsiveness induced by anti-CD40 ligand monoclonal antibody. Animals that received anti-CD40 ligand monoclonal antibody demonstrated robust regeneration across nerve allografts, similar to that seen in the autograft control in the contralateral arm. The histomorphometric analysis of allografts in the untreated animals demonstrated significantly worse measurements compared with their matched autograft controls. Animals that received anti-CD40 ligand monoclonal antibody with concomitant skin allografts had virtually no evidence of nerve regeneration through allografts. Allogeneic skin and nerve allografts applied 2 to 12 months after withdrawal of anti-CD40 ligand monoclonal antibody therapy were consistently rejected. This study demonstrates that anti-CD40 ligand monoclonal antibody prevents rejection and allows regeneration of peripheral nerve allografts in nonhuman primates. The effect of anti-CD40 ligand monoclonal antibody appears to be transient, however, with restoration of immunocompetence shortly after withdrawal of therapy.     

Relationship between the blood flow in a spinal nerve root and its functional state in patients with lumbar disk hernia

Capillary blood flow in the spinal roots was studied in 52 patients with lumbar osteochondrosis during discectomy for lumbar disk herniation, before and after decompression. It was determined that the value of the capillary blood flow in the nerve root influenced markedly the severity of the pain syndrome, the threshold of sensitivity to pain in the dermatome involved, and the strength of the indicator group of muscles. It was shown that the level of intact reserves of the spinal nerve root microcirculation influenced the degree of functional restoration after the discectomy.     

Changes in the skin epithelium and nerve elements of the rat sole with directed regeneration of the sciatic nerve

The work has been performed on Wistar rats and non-inbred animals. Their ischiatic nerves have been dissected at the femoral superior third under nembutal narcosis. The end of the sectioned nerve are connected by a fragment of an aorta from rats of the same age. The state of nervous elements and dermal epithelium of the hind extremity sole in the animals is studied by means of general histological and neurohistological techniques. Mitotic activity of cells in the plantar epidermis, thickness as a whole and its separate layers are estimated, keratinization coefficient and correlation of thickness of separate sheaths in the whole layer are calculated. Use of the arterial vessels for connecting the end of the cut ischiatic nerve, trophic ulcers, that usually take place after the nerve section, do not develop. At early stages after the operation mitotic activity in the epidermis decreases by 70%, and the layer thickness--by 40%. Restoration of both indices proceeds slowly. As soon as the regenerating nerve fibers reach the distal part of the ischiatic nerve, the state of the epidermis improves: the mitotic activity differs from the normal by 20-30%, and thickness of the epithelium--by 28-30%. Coordination of thickness of separate layers in the epidermis is not nearly disturbed. It remains in the same state up to complete restoration of receptory structures in the rat plantar skin (during 9-9.5 months after the operation).     

Activity cage as a method to analyze functional recovery after sciatic nerve injury in mice

The aim of this paper is to show the activity cage as a viable method for tracking functional nerve recovery. The activity cage measures spontaneous coordinate activity, meaning movement in either the horizontal or vertical plane, of experimental animals within a specified amount of time. This uses a minimum of researcher time conducting functional testing to determine functional recovery of the nerve. Using microsurgical forceps, a crush injury was inflicted unilaterally, on the left side, upon the 4-month-old C3H mice creating a very high degree of pressure for 6 s upon the exposed sciatic nerve. The locomotion function of the mice was evaluated using the activity cage preoperatively, 1, 7, 14, 21, and 28 days after the surgical procedure. We found that using the activity cage functional recovery occurred by 14 days after nerve crush injury. It was also shown that, coinciding with functional recovery, immunohistochemistry changes for GD1a and nNOS appeared at the level of L4, where the sciatic nerve joins the spinal column. GD1a and nNOS have both been linked to regenerative processes in mammalian nervous systems.     

Activity cage as a method to analyze functional recovery after sciatic nerve injury in mice

The aim of this paper is to show the activity cage as a viable method for tracking functional nerve recovery. The activity cage measures spontaneous coordinate activity, meaning movement in either the horizontal or vertical plane, of experimental animals within a specified amount of time. This uses a minimum of researcher time conducting functional testing to determine functional recovery of the nerve. Using microsurgical forceps, a crush injury was inflicted unilaterally, on the left side, upon the 4-month-old C3H mice creating a very high degree of pressure for 6 s upon the exposed sciatic nerve. The locomotion function of the mice was evaluated using the activity cage preoperatively, 1, 7, 14, 21, and 28 days after the surgical procedure. We found that using the activity cage functional recovery occurred by 14 days after nerve crush injury. It was also shown that, coinciding with functional recovery, immunohistochemistry changes for GD1a and nNOS appeared at the level of L4, where the sciatic nerve joins the spinal column. GD1a and nNOS have both been linked to regenerative processes in mammalian nervous systems.     

Melanocytes: morphological basis for an exteroceptive sensory system for monitoring ultraviolet radiation

From the conventional medical perspective, melanocytes have been traditionally viewed as epidermal responders/reactors to ultraviolet radiation. In this paper we have begun an analysis of the functional significance of melanocytes as monitors for ultraviolet radiation with their neuronal, endocrinological and immunological intercalations. This preliminary study was performed using as a human model unilateral sural nerve sensory blockade before and after ultraviolet exposure to both feet while the unblocked foot acted as the control. Full thickness surgical skin biopsies were taken of (I), normal pre-exposure skin, in the center of the two centimeter exposure area of both the sural nerve blocked area (II-III) and control (IV-V) one centimeter outside the exposed area in both the sural nerve blocked foot and the control or non-blocked foot. The objective clinical, blinded morphological and immuno-histochemical data from this experiment support the initial conclusion that neuronal connection is necessary for the normal ultraviolet exposure dermal reaction. Based upon this study, we further propose the existence of an exteroceptive sensory system in which melanocytes, with direct nervous system connection initiate an ultraviolet radiation reactive response that mobilizes both conventional endocrine and immune pathways.     

Merkel cell-nerve cell interaction undergoes formation of a synapse-like structure in a primary culture

Merkel cells have been assumed to guide nerve fibers to the skin. However, there has been little in vitro evidence that supports this hypothesis, because there is no suitable established culture system of Merkel cells. Here we show that Merkel cells isolated from rat footpad skin were successfully cultured in a monolayer with keratinocytes. Keratinocytes did not affect any structural changes in Merkel cells. When nerve cells (NG108-15 or PC12) were added to the culture system, both nerve fibers and cytoplasmic processes of Merkel cells outgrew and cooperatively organized synapse-like structures at their contact points. Nerve cells promoted Merkel cell survival, compared with keratinocytes only. Merkel cell proliferation was not detected in all conditions, even with nerve growth factor, neurotrophin-3, interleukin-6 and tumor necrosis factor-alpha. The data suggest, firstly, that Merkel cells may guide nerve fibers to the skin by interacting with nerve cells; and, secondly, that nerve cells, but not keratinocytes, may produce some survival factors other than the cytokines above for Merkel cells, although Merkel cells may be a terminally differentiated cell type. Our method could open a way to study Merkel cell biology.     

Ecological indicators for assessing ecological success of forest restoration: a world review

Restoration is increasingly being used to reverse degradation and destruction of forest ecosystems. With increasing investment in restoration, there is an urgent need to develop effective programs to assess treatment efficacy and effects. We conducted a global review of forest restoration assessments, in order to identify geographic trends in the locations where assessments have been implemented and the specific ecological attributes (ecosystem composition, structure, and function) and indicators being used to measure effects. We found that the number of forest restoration assessments varied by region and was not related to degree of degradation or restoration need. Some regions, like Africa, which have experienced high rates of forest loss and degradation, had few assessments. The majority (43%) of assessments included indicators for only two of three key ecological attributes (composition‐structure or composition‐function) and assessments on average used fewer than three indicators per attribute. The most commonly employed indicators for composition were richness and abundance of plant species and for structure were height and diameter of trees, variables that are generally relatively easy to measure. The use of functional indicators has been increasing over time and they are now more commonly used than structural indicators. The most common functional indicators were soil functions. Most investigators evaluated treatment effects for 6–10 years after implementation. Our findings related to gaps in analysis of ecological indicators can serve as a guide for developing monitoring and assessment protocols for current global forest restoration initiatives by 2020–2030.     

Mesenteric and tactile Pacinian corpuscles are anatomically and physiologically comparable

Pacinian corpuscles (PCs) in cat mesentery have been studied extensively to help determine the structural and functional bases of tactile mechanotransduction. Although we, like many other investigators, have found that the mesenteric receptors are anatomically very similar to those found in mammalian skin, few physiological characteristics of the mesenteric PCs and those of the skin have been compared. Action-potential rate-amplitude and frequency characteristics (10 Hz-1 KHz), as well as interval (IH) and peri-stimulus-time (PSTH) histograms in response to sinusoidal displacements were obtained from nerve fibers innervating mesenteric PCs and from PC fibers innervating cat glabrous skin. The intensity characteristics obtained on both preparations showed similar response profiles, including equal slopes for low stimulus intensities (approximately 10, with impulse ratios/20 dB displacement) and one and two impulse/cycle entrainment. The frequency characteristics of both groups were U-shaped with similar low-frequency slopes (-12.5 dB/octave) and bandwidths (Q(3dB) = 1.4). The best frequency for both the tactile PCs' and mesenteric PCs was 250 Hz, which is in the expected range. The IHs showed entrainment and the PSTHs showed neither transient responses nor adaptation to steady-state sinusoidal stimuli. The functional similarity between mesenteric PCs' nerve responses and those of tactile PC afferents, as well as the receptors' anatomical similarity, lead us to suggest that the mesenteric PC can act as a model for those in the skin. Furthermore, since the frequency characteristics of the two PC types are similar, it is concluded that the skin, while attenuating stimulus intensity, does not impart temporal filtering of vibratory stimuli.     

Mesenteric and tactile Pacinian corpuscles are anatomically and physiologically comparable

Pacinian corpuscles (PCs) in cat mesentery have been studied extensively to help determine the structural and functional bases of tactile mechanotransduction. Although we, like many other investigators, have found that the mesenteric receptors are anatomically very similar to those found in mammalian skin, few physiological characteristics of the mesenteric PCs and those of the skin have been compared. Action-potential rate-amplitude and frequency characteristics (10 Hz–1 KHz), as well as interval (IH) and peri-stimulus-time (PSTH) histograms in response to sinusoidal displacements were obtained from nerve fibers innervating mesenteric PCs and from PC fibers innervating cat glabrous skin. The intensity characteristics obtained on both preparations showed similar response profiles, including equal slopes for low stimulus intensities (approximately 10, with impulse ratios/20 dB displacement) and one and two impulse/cycle entrainment. The frequency characteristics of both groups were U-shaped with similar low-frequency slopes (?12.5 dB/octave) and bandwidths (Q_3dB = 1.4). The best frequency for both the tactile PCs' and mesenteric PCs was 250 Hz, which is in the expected range. The IHs showed entrainment and the PSTHs showed neither transient responses nor adaptation to steady-state sinusoidal stimuli. The functional similarity between mesenteric PCs' nerve responses and those of tactile PC afferents, as well as the receptors’ anatomical similarity, lead us to suggest that the mesenteric PC can act as a model for those in the skin. Furthermore, since the frequency characteristics of the two PC types are similar, it is concluded that the skin, while attenuating stimulus intensity, does not impart temporal filtering of vibratory stimuli.     

A nanofibrous PHBV tube with Schwann cell as artificial nerve graft contributing to Rat sciatic nerve regeneration across a 30-mm defect bridge

Abstract

A nanofibrous PHBV nerve conduit has been used to evaluate its efficiency based on the promotion of nerve regeneration in rats. The designed conduits were investigated by physical, mechanical and microscopic analyses. The conduits were implanted into a 30-mm gap in the sciatic nerves of the rats. Four months after surgery, the regenerated nerves were evaluated by macroscopic assessments and histology. This polymeric conduit had sufficiently high mechanical properties to serve as a nerve guide. The results demonstrated that in the nanofibrous graft with cells, the sciatic nerve trunk had been reconstructed with restoration of nerve continuity and formatted nerve fibers with myelination. For the grafts especially the nanofibrous conduits with cells, muscle cells of gastrocnemius on the operated side were uniform in their size and structures. This study proves the feasibility of artificial conduit with Schwann cells for nerve regeneration by bridging a longer defect in a rat model.     

Behavioral plasticity and central regeneration of locomotor reflexes in the freshwater oligochaete, Lumbriculus variegatus. I: Transection studies

Abstract. Access to the ventral nerve cord in living specimens of Lumbriculus variegatus , an aquatic oligochaete, is normally impossible because surgical invasion induces segmental autotomy (self-fragmentation). We show here that nicotine is a powerful paralytic agent that reversibly immobilizes worms, blocks segmental autotomy, and allows experimental access to the nerve cord. Using nicotine-treated worms, we transected the ventral nerve cord and used non-invasive electrophysiological recordings and behavioral analyses to characterize the functional recovery of giant nerve fibers and other reflex pathways. Initially, after transection, medial giant fiber (MGF) and lateral giant fiber (LGF) spikes conducted up to, but not across, the transection site. Reestablishment of MGF and LGF through-conduction across the transection site occurred as early as 10 h (usually by 20 h) after transection. Analyses of non-giant-mediated behavioral responses (i.e., helical swimming and body reversal) were also made following nerve cord transection. Immediately after transection, functional reorganization of touch-evoked locomotor reflexes occurred, so that the two portions of the worm anterior and posterior to the transection site were independently capable of helical swimming and body reversal responses. Similar reorganization of responses occurred in amputated body fragments. Reversion back to the original whole-body pattern of swimming and reversal occurred as early as 8 h after transection. Thus, functional restoration of the non-giant central pathways appeared slightly faster than giant fiber pathways. The results demonstrate the remarkable plasticity of locomotor reflex behaviors immediately after nerve cord transection or segment amputation. They also demonstrate the exceptional speed and specificity of regeneration of the central pathways that mediate locomotor reflexes.     

Sensory reinnervation in microsurgical reconstruction of the heel

Six sensory reinnervation techniques were carried out in 10 patients who underwent reconstruction of the weight-bearing surface of the heel by microsurgical free-tissue transfer. The techniques include the use of neurovascular island flaps, neurosensory flaps, sensory nerve grafts to skin flaps, coaptation of the sensory nerve to the motor nerve of the muscle flaps, direct sensory nerve transfer, and sensory nerve graft transfer. In all patients, some sensation developed, characterized by sensation to light touch, to dull objects, to pinprick, to pain, and to tickling. Three patients developed the ability to distinguish sharp from dull objects and the sensation of pain. The remaining seven had the sensation of touch to various mechanical stimuli. In nine patients, the sensation is located in the weight-bearing surface of the reconstructed heel. Five patients bear weight on the reconstructed surface at least 6 hours per day. Three participate actively in sports. Split-thickness skin graft-muscle flaps were more prone to breakdown than skin flaps. Full-thickness skin flaps appear necessary for the production of pain sensation and the more discriminating sensations. Preliminary results suggest a functional benefit after sensory reinnervation.     

Effect of reinnervation on the state of transplanted kidneys in dogs]

In the experiments lasted for 2 years, the effect of surgical reinnervation on the morphofunctional state of autotransplanted and denervated kidney was studied in 175 dogs. General morphological, impregnational, special neurohistological investigations and kidney scanning demonstrated that 2 months after surgical reinnervation of the autotransplantated and denervated kidneys, active sprouting of nerve fibers, adrenergic and cholinergic including, was started along the degenerated nerve trunks, and by the 4th month the nerve fibers reached the renal glomeruli, tubules and pelves. A rather complete restoration of the organic innervation resembling that of the intact organ provided stability of the general morphological structure in the reinnervated kidney, its functional ability and prevented atrophy, sclerosis and stable functional depression observed in cases of renal transplantation and denervation without surgical reinnervation. From this point of view, the problem of necessary active surgical reinnervation of the renal transplantation is discussed.     

Influence of sensory peptidergic innervation on human skin blood flow oscillations in the range 0.047–0.069 Hz

Thirty-six healthy subjects and 65 patients with neurogenic inflammation (complex regional pain syndrome of the hand) or denervation syndromes (after median and ulnar nerve injuries or transplantation of denervated vascularized musculocutaneous autografts), as well as after thoracoscopic sympathectomy, underwent laser Doppler flowmetry with spectral wavelet analysis of the blood flow oscillations in cutaneous microvessels and thermography. It was shown that, along with maintenance of the blood flow oscillations of endothelial genesis, peptidergic sensory nerve fibers (SPFs) are involved in activating independent, including high-amplitude, oscillations in the myogenic range 0.047–0.069 Hz (an average of three to four oscillations per minute). The above-mentioned oscillations were recorded against the background of neurogenic inflammation and nociceptive activation of C afferents after nerve injuries, as well as in the course of functional tests in healthy subjects (the forearm skin electrostimulation test, capsaicin application). Sympathectomy and hyposympathicotonia contributed to their manifestation; they were not detected under the conditions of severe sensory-trophic skin denervation. The appearance of high-amplitude blood flow oscillations in human skin microvessels at a frequency of 0.047–0.069 Hz may serve as an objective criterion of SPF activation.     

Equilibria of frog nerve with different external concentrations of sodium ions

Using the ability of the nerve fibers to conduct impulses as indicator of changes in the concentration of sodium ions in the interstitial spaces of nerve an evaluation has been made of the diffusion constant of sodium ions. The calculated minimal value (0.62 x 10(-4) cm.(2)/min.) undoubtedly is much too low; nevertheless, it is still so high that as a rule the diffusion of sodium ions is far more rapid than the establishment of excitability changes; therefore, diffusion times need not be taken into account in the interpretation of ordinary experiments. By measurements of the changes in the longitudinal conductivity of nerve which result from changes in the external concentration of sodium chloride an evaluation has been made of the diffusion constant of sodium chloride in the interstitial spaces of nerve. A minimal value for this constant is 1.4 x 10(-4) cm.(2)/min. The evidence presented would be compatible with the assumption that the permeability of the connective tissue sheath for sodium ions decreases slightly after the concentration of sodium ions in the interstitial spaces of the nerve has become negligible; the evidence, however, shows that changes in the permeability of the sheath cannot play a significant role in determining the temporal courses of the development of inexcitability in a sodium-free medium and of the restoration of excitability by added sodium ions. If a decrease in the permeability of the sheath should take place in a sodium-free medium, the change would be small and would occur after the nerve fibers have become inexcitable; on the other hand the action of a moderate concentration of sodium ions would be sufficient to restore the permeability of the sheath. As measured by the recovery by A fibers of the ability to conduct impulses the restoration by 0.1 N sodium ions of nerve that has been deprived of sodium for 15 to 20 hours, i.e. for several hours after the nerve fibers have become inexcitable, begins after a significant delay, since no A fiber begins to conduct impulses in less than 8 or 10 minutes. The delay is referable to the fact that, before the A fibers can regain the ability to conduct impulses, those changes in their properties have to be reversed, which have taken place in the absence of sodium ions. Usually within 1 minute after sodium ions are made available to the nerve the polarizability of the membrane by the anodal current begins to increase; the A fibers soon begin to produce unconducted impulses in response to the break of the anodal current; then, they produce unconducted impulses in response to the closure of the cathodal current, and finally they become able to conduct impulses, although at a markedly reduced speed. The C fibers, that become inexcitable in a sodium-free medium later than the A fibers, begin to conduct impulses within 1 minute or 2 after 0.1 N sodium ions are made available to the nerve. Treatment of a nerve, that has been kept in a sodium-free medium, for 15 to 20 hours, with a moderate concentration of sodium ions (0.015, 0.02 N), acting for 1 hour or 2, is not sufficient to restore the ability to conduct impulses to more than a few A fibers, but it produces in a relatively large number of fibers a partial restoration, so that when the concentration of sodium ions outside the epineurium is increased by 0.005 or 0.01 N a significant number of A fibers begin to conduct impulses within less than 5 seconds. Initially the recovery progresses with great rapidity, but after a small number of minutes the height of the conducted spike remains practically stationary. Increase of the external concentration of sodium ions by a small amount again causes a rapid enhancement of the recovery, but once more, after a few minutes the height of the spike remains practically stationary, etc. A subnormal concentration of sodium ions may restore to all the A fibers the ability to conduct impulses, but only 0.1 N sodium ions are able to produce a complete restoration of the speed of conduction, and only after they have been allowed to act for a considerable period of time. The ability of all the C fibers to conduct impulses may be restored by relatively small concentrations of sodium ions, 0.02 to 0.025 N. Nerve fibers that have become inexcitable in a sodium-free medium and have been restored by sodium ions are far more sensitive to the effect of the lack of sodium than the fibers of untreated nerve. Repeated removal and addition of sodium ions may bring the nerve fibers, especially those of spinal roots, to a state in which the sensitivity to the lack of sodium is exceedingly great; spinal root fibers may then begin to become inexcitable in a sodium-free medium within a few seconds. Treatment of the nerve with 0.1 N sodium ions for 1 hour or 2 is sufficient to bring about a marked increase in the resistance to the lack of sodium. On the other hand keeping a nerve in Ringer's solution or in the presence of 0.04 N sodium ions does not produce a readily detectable increase in the sensitivity to the lack of sodium. Even the resistance of nerve kept in the presence of 0.025 N sodium ions for 23 hours is very high, since after 2 hours in a sodium-free medium more than two-thirds of the initially conducting fibers will be able to conduct impulses. Frog nerve reaches different states of equilibrium with different external concentrations of sodium ions. The states are characterized by the degree of effectiveness of the nerve reaction, the speed of conduction of impulses, and the number of conducting fibers. Approximately the same equilibrium state may be reached by (a) leaving the nerve for 20 to 24 hours in the presence of a subnormal concentration of sodium ions and (b) by leaving the nerve in a sodium-free medium for 15 to 20 hours, restoring it with 0.1 N sodium ions acting for a short period of time, rendering it inexcitable again in a sodium-free medium, and finally restoring it with a moderate concentration of sodium ions. If, however, the nerve that has been kept in a sodium-free medium for 15 to 20 hours is restored directly by a moderate concentration of sodium ions the state will not be reached, at least not for several hours, which corresponds to equilibrium with that concentration. The role of sodium in nerve physiology is discussed. Sodium participates in at least four processes, (a) The regulation of the concentration of water outside the nerve fibers; (b) the regulation of the total value of the membrane potential; (c) the production of the nerve impulse, and (d) the establishment of the nerve reaction. In so far as processes (c) and (d) are concerned only the sodium present inside the nerve fibers plays a role; the presence of sodium ions outside the nerve fibers is important only because in the absence of interstitial sodium ions the nerve fibers lose a part of their internal sodium content. The nerve impulse and the nerve reaction may be produced for long periods of time after the concentration of sodium ions outside the nerve fibers has become negligible. A working hypothesis is put forward according to which the internal sodium content and the interstitial concentration of sodium ions are in equilibrium in so far as a different internal sodium content corresponds to each interstitial concentration. The properties of the nerve fibers are determined by the internal sodium content. The change in properties, i.e. in the state of the nerve fibers, results from processes that take place inside the nerve fibers after the interstitial concentration of sodium ions and consequently also the internal sodium content have been changed.