Immunohistochemical Femoral Nerve Study Following Bisphosphonates Administration.

Background and objectives: Bisphosphonates represent selective inhibitors of excess osteoblastic bone resorption that characterizes all osteopathies, targeting osteoclasts and their precursors. Their long-term administration in postmenopausal women suffering from osteoporosis has resulted in neural adverse effects. The current study focuses on the research of possible alterations in the femoral nerve, caused by bisphosphonates. We hypothesized that bisphosphonates, taken orally (per os), may produce degenerative changes to the femoral nerve, affecting lower-limb posture and walking neuronal commands. Materials and Methods: In order to support our hypothesis, femoral nerve specimens were extracted from ten female 12-month-old Wistar rats given 0.05 milligrams (mg) per kilogram (kg) of body weight (b.w.) per week alendronate per os for 13 weeks and from ten female 12-month-old Wistar rats given normal saline that were used as a control group. Specimens were studied using immunohistochemistry for selected antibodies NeuN (Neuronal Nuclear Protein), a protein located within mature, postmitotic neural nucleus, and cytosol and Sox10 (Sex-determining Region Y (SRY) - High-Motility Group (HMG) - box 10). The latter marker is fundamental for myelination of peripheral nerves. Obtained slides were examined under a light microscope. Results: Samples extracted from rats given alendronate were more Sox10 positive compared to samples of the control group, where the marker's expression was not so intense. Both groups were equally NeuN positive. Our results are in agreement with previous studies conducted under a transmission electron microscope. Conclusions: The suggested pathophysiological mechanism linked to histological alterations described above is possibly related to toxic drug effects on Schwann and neuronal cells. Our hypothesis enhances the existing scientific evidence of degenerative changes present on femoral nerve following bisphosphonates administration, indicating a possible relationship between alendronate use and neuronal function.

Low back pain due to superior cluneal nerve entrapment: A clinicopathologic study.

INTRODUCTION: We studied the clinical and nerve pathologic features in 6 patients whose low back pain (LBP) was relieved by superior cluneal nerve (SCN) neurectomy to determine whether nerve compression was the mechanism underlying this type of LBP. METHODS: All 6 patients (7 nerves) underwent SCN neurectomy for intractable LBP. Their clinical outcomes and the pathologic features of 7 nerves were reviewed. RESULTS: All patients reported LBP relief immediately after SCN neurectomy. Pathologic study of the 7 resected nerves showed marked enlargement, decreased myelinated fiber density, an increase in thinly myelinated fibers (n = 2), perineurial thickening (n = 5), subperineurial edema (n = 4), and Renaut bodies (n = 4). At the distal end of 1 enlarged nerve, we observed a moderate reduction in the density and marked reduction in the number of large myelinated fibers. DISCUSSION: The pathologic findings and effectiveness of neurectomy suggest that, in our patients, SCN neuropathy likely elicited LBP via nerve compression. Muscle Nerve 57: 777-783, 2018.

Alpha-synuclein facilitates to form short unconventional microtubules that have a unique function in the axonal transport.

Although α-synuclein (αSyn) has been linked to Parkinson's disease (PD), the mechanisms underlying the causative role in PD remain unclear. We previously proposed a model for a transportable microtubule (tMT), in which dynein is anchored to a short tMT by LIS1 followed by the kinesin-dependent anterograde transport; however the mechanisms that produce tMTs have not been determined. Our in vitro investigations of microtubule (MT) dynamics revealed that αSyn facilitates the formation of short MTs and preferentially binds to MTs carrying 14 protofilaments (pfs). Live-cell imaging showed that αSyn co-transported with dynein and mobile ßIII-tubulin fragments in the anterograde transport. Furthermore, bi-directional axonal transports are severely affected in αSyn and γSyn depleted dorsal root ganglion neurons. SR-PALM analyses further revealed the fibrous co-localization of αSyn, dynein and ßIII-tubulin in axons. More importantly, 14-pfs MTs have been found in rat femoral nerve tissue, and they increased approximately 19 fold the control in quantify upon nerve ligation, indicating the unconventional MTs are mobile. Our findings indicate that αSyn facilitates to form short, mobile tMTs that play an important role in the axonal transport. This unexpected and intriguing discovery related to axonal transport provides new insight on the pathogenesis of PD.

Effects of metabolic syndrome on the ultrastructure of the femoral nerve in aging rats.

The aim of the present study was to characterize the morphometry of the femoral nerve in aging rats with metabolic syndrome compared to controls. Systolic blood pressure and fasting plasma glucose were measured, and myelinated and unmyelinated fibers in the femoral nerves were quantitatively assessed under electron microscopy. Aging rats exposed to a regimen of metabolic syndrome developed elevation of plasma glucose concentration, mild hypertension and polyneuropathy characterized by a decrease in myelin fiber area, axon diameter, myelin sheath thickness and myelin fiber loss in the femoral nerve. The histogram of size distribution for myelinated fibers and axons from the aging rats of the control group was bimodal. For aging MS animals, the histogram turned out to be unimodal. The ultrastructure of unmyelinated fibers and of Schwann cells in 18-month-old rats was well preserved. Granules of lipofuscin were seen in unmyelinated fiber axons of 18-month-old rats with MS. The damage percentage of the large myelinated fibers has increased significantly in 18-month-old and 18-month-old (MS) rats in relation to the controls. No significant difference was observed among the groups for the g-ratio. Comparing the three groups, the number of neurotubules and neurofilaments in myelinated fibers of 18-month-old rats with MS was significantly smaller than for the groups of 18-month-old and 14-month-old rats. The overall changes seen in the femoral nerve from aging rats seem minor compared to the changes in the aging rats with MS, suggesting that long-term MS accelerates the progressive modifications in peripheral nerves that develop in old age.

TACE (ADAM17) inhibits Schwann cell myelination.

Tumor necrosis factor-α-converting enzyme (TACE; also known as ADAM17) is a proteolytic sheddase that is responsible for the cleavage of several membrane-bound molecules. We report that TACE cleaves neuregulin-1 (NRG1) type III in the epidermal growth factor domain, probably inactivating it (as assessed by deficient activation of the phosphatidylinositol-3-OH kinase pathway), and thereby negatively regulating peripheral nervous system (PNS) myelination. Lentivirus-mediated knockdown of TACE in vitro in dorsal root ganglia neurons accelerates the onset of myelination and results in hypermyelination. In agreement, motor neurons of conditional knockout mice lacking TACE specifically in these cells are significantly hypermyelinated, and small-caliber fibers are aberrantly myelinated. Further, reduced TACE activity rescues hypomyelination in NRG1 type III haploinsufficient mice in vivo. We also show that the inhibitory effect of TACE is neuron-autonomous, as Schwann cells lacking TACE elaborate myelin of normal thickness. Thus, TACE is a modulator of NRG1 type III activity and is a negative regulator of myelination in the PNS.

Systematic evaluation of the highest current threshold for regional anaesthesia in a porcine model.

BACKGROUND: The purpose of this study was to determine systematically the highest minimal stimulation current threshold for regional anaesthesia in pigs. METHODS: In an established pig model for regional anaesthesia, needle placements applying electric nerve stimulation were performed. The primary outcome was the frequency of close needle to nerve placements as assessed by resin injects and subsequent anatomical evaluation. Following a statistical model (continual reassessment method), the applied output currents were selected to limit the necessary number of punctures, while providing guidance towards the highest output current range. RESULTS: Altogether 186 punctures were performed in 11 pigs. Within the range of 0.3-1.4 mA, no distant needle to nerve placement was found. In the range of 1.5-4.1 mA, 43 distant needle to nerve placements occurred. The range of 1.2-1.4 mA was the highest interval that resulted in a close needle to nerve placement rate of > or =95%. CONCLUSIONS: In the range of 0.3-1.4 mA, all resin deposition was found to be adjacent to nerve epineurium. The application of minimal current intensities up to 1.4 mA does not obviously lead to a reduction of epineural injectate contacts in pigs. These findings suggest that stimulation current thresholds up to 1.4 mA result in equivalent needle tip localisation in pigs.

Impact of vascularization type on peripheral nerve microstructure.

Experimental and clinical studies have demonstrated that vascularized nerve grafts are superior to nonvascularized nerve grafts with respect to healing. By means of the inherent vascularity in vascularized nerve grafts, Schwann cells remain viable, and endoneurial necrosis and fibrosis are not seen. In this study the effects of three different vascularization patterns on the vascular microstructure of a nerve segment in the rat based on the femoral artery and vein was investigated. Sixty adult male Wistar Albino rats were divided into five groups. In each group, a 1.5-cm segment of femoral nerve was transected at two sides, without disturbing the unity of the contents of the femoral sheath. The experimental design consisted of prefabricated venous nerve segment, venous nerve segment, arterial nerve segment, no blood flow, and controls groups. To assess the microstructure of the nerve segment, myelin and Schwann cell morphology and fibrosis were examined. There were many Schwann cells with near normal morphology in the venous nerve segment and arterial nerve segment groups. In conclusion, the venous nerve segment model in which Schwann cell viability was high due to the presence of sufficient and uninterrupted blood supply to the nerve graft, resulting in successful nerve healing, showed superior results over others.

Altered ion channels in an animal model of Charcot-Marie-Tooth disease type IA.

How demyelination and remyelination affect the function of myelinated axons is a fundamental aspect of demyelinating diseases. We examined this issue in Trembler-J mice, a genetically authentic model of a dominantly inherited demyelinating neuropathy of humans. The K+ channels Kv1.1 and Kv1.2 channels were often improperly located in the paranodal axon membrane, typically associated with improperly formed paranodes, and in unmyelinated segments between internodes. As in wild-type nerves, Trembler-J nodes contained Nav1.6, ankyrin-G, betaIV-spectrin, and KCNQ2, but, unlike wild-type nerves, they also contained Kv3.1b and Nav1.8. In unmyelinated segments bordered by myelin sheaths, these proteins were clustered in heminodes and did not appear to be diffusely localized in the unmyelinated segments themselves. Nodes and heminodes were contacted by Schwann cells processes that did not have the ultrastructural or molecular characteristics of mature microvilli. Despite the presence of Nav1.8, a tetrodotoxin-resistant sodium channel, sciatic nerve conduction was at least as sensitive to tetrodotoxin in Trembler-J nerves as in wild-type nerves. Thus, the profound reorganization of axonal ion channels and the aberrant expression of novel ion channels likely contribute to the altered conduction in Trembler-J nerves.

The changes of peripheral nerve microstructure after surgical manipulation--experiment on rat model.

The aim of our study was to describe histopathology of the peripheral nerve after its circular release followed by embedding in different environs. We operated on 18 male rats divided into 3 groups. In the first group right femoral nerve was surgically released. In the second group the nerve was enveloped by the subcutaneous fat flap. In the third one the nerve was wrapped up by the skeletal muscle. Six weeks later the animals were killed by exsanguination. The femoral nerve, in the first group, did not show any pathological changes. In the second group 3 animals appeared normal or nearly normal, nevertheless in 3 of them perineural fibrosis and axonal degeneration were observed. Histological reaction in the third group disclosed dispersed axonal injury. Our experiments using rat model imitate situation in humans. The results obtained will help us in making meaningful decision when performing peripheral nerve injury.

The role of macrophages in demyelinating peripheral nervous system of mice heterozygously deficient in p0.

Mice heterozygously deficient in the p0 gene (P0(+/-)) are animal models for some forms of inherited neuropathies. They display a progressive demyelinating phenotype in motor nerves, accompanied by mild infiltration of lymphocytes and increase in macrophages. We have shown previously that the T lymphocytes are instrumental in the demyelination process. This study addresses the functional role of the macrophage in this monogenic myelin disorder. In motor nerves of P0(+/)- mice, the number of macrophages in demyelinated peripheral nerves was increased by a factor of five when compared with motor nerves of wild-type mice. Immunoelectron microscopy, using a specific marker for mouse macrophages, displayed macrophages not only in the endoneurium of the myelin mutants, but also within endoneurial tubes, suggesting an active role in demyelination. To elucidate the roles of the macrophages, we crossbred the myelin mutants with a spontaneous mouse mutant deficient in macrophage colony-stimulating factor (M-CSF), hence displaying impaired macrophage activation. In the P0-deficient double mutants also deficient in M-CSF, the numbers of macrophages were not elevated in the demyelinating motor nerves and demyelination was less severe. These findings demonstrate an active role of macrophages during pathogenesis of inherited demyelination with putative impact on future treatment strategies.

Intraneural stimulation elicits an increase in subcutaneous interstitial glycerol levels in humans.

1. The effect of intraneural electrical stimulation of the lateral femoral cutaneous nerve on lipolysis in the innervation territory of the stimulated nerve fascicle was studied in seven healthy women. Lipolysis was evaluated by microdialytic measurement of the interstitial glycerol concentration in subcutaneous adipose tissue. 2. Ten minutes of unilateral intraneural stimulation elicited a 22 +/- 8 % (mean +/- s.e.m.) increase in glycerol levels in the stimulated region (P < 0.05), whereas no change was registered in the corresponding area of the contralateral unstimulated leg. 3. Significantly higher glycerol levels in the stimulated vs. contralateral unstimulated region (47 +/- 13 %, P < 0.05) were already observed at baseline (30 min resting period preceding the 10 min stimulation), in all probability as a consequence of the nerve searching procedure and trial stimulations. After the 10 min stimulation, the overall glycerol increase was 72 +/- 17 % compared with the contralateral leg, illustrating the degree of lipolysis induced by the whole experimental procedure. 4. The sympathetic discharge in the lateral femoral nerve (6 recordings) showed typical characteristics of skin sympathetic activity, and the firing pattern was strikingly similar to simultaneously recorded sympathetic discharge in cutaneous nerve fascicles innervating regions without prominent subcutaneous fat stores (2 double nerve recordings). Thus, no component of cutaneous sympathetic outflow specific for the nerve innervating prominent subcutaneous fat stores could be identified. 5. Our findings suggest that sympathetic nerve fibres travelling in cutaneous nerve fascicles exert a regulatory influence on subcutaneous fat tissue in humans. The combination of intraneural recording/stimulation and subcutaneous microdialysis provides a model for evaluating neural control of human fat metabolism.

Subtle roles of neural cell adhesion molecule and myelin-associated glycoprotein during Schwann cell spiralling in P0-deficient mice.

Peripheral nerves of P0-deficient mice display a severe dysmyelinating phenotype, confirming the view that P0 mediates myelin formation and compaction. In addition to the compromised myelin organization, an elevated expression of several cell recognition molecules was described in the axon-Schwann cell units of P0-deficient mice. The present study was performed to focus on the subcellular localization and functional roles of two of these up-regulated molecules, the neural cell adhesion molecule (N-CAM) and the myelin-associated glycoprotein (MAG). We show by postembedding immunoelectron microscopy that in peripheral nerves of P0-deficient mice both molecules are expressed in noncompacted myelin-like regions. In addition, N-CAM, but not MAG, is detected in partially compacted myelin. By the generation of P0/N-CAM- and P0/MAG-deficient double mutants, we investigated the roles of the dysregulated molecules in P0-deficient mice. In 4-week-old double mutants, the dysmyelinating phenotype of the axon-Schwann cell units was very similar to that seen in the P0-deficient single mutants, suggesting that neither N-CAM nor MAG are responsible for the poor myelin compaction in P0-deficient mice. However, the noncompacted turns surrounding the abnormally compacted regions were significantly reduced in number in P0/MAG mutants as compared to P0 or N-CAM/P0 mice. During formation of myelin sheaths, absence of N-CAM resulted in a transient retardation of Schwann cell spiralling in P0-deficient mice, whereas absence of MAG impaired Schwann cell spiralling for a more extended time period. Our findings demonstrate for the first time that MAG and also N-CAM can play significant roles during myelin formation in the peripheral nervous system. Because these functional roles are detectable only in the absence of P0, our results confirm the view that myelin-related molecules can play distinct, but also partially overlapping roles.

Myelin formation by Schwann cells in the absence of beta4 integrin.

The interaction of the Schwann cell with its basal lamina has been hypothesized to be an important prerequisite for the formation of a myelin sheath in the peripheral nervous system. One possible player in this interaction is beta4 integrin; it is up-regulated during myelin formation and, in association with alpha6 integrin, can interact with particular components of the Schwann cell basal lamina. In order to characterize the functional roles of beta4 integrin during myelination, we investigated myelination in the absence of beta4 integrin, i.e., in peripheral nerve tissue from beta4 integrin-deficient mice. Because the mutants die within several hours after birth, we cultured dorsal root ganglia from neonatal mutants under conditions that promote myelination, quantified the myelin segments by immunofluorescence, and investigated the ultrastructure of the cultured myelin sheaths. In another approach, we quantified the few myelin sheaths that are detectable in femoral nerves of newborn animals. Based on both approaches, we conclude that myelination by Schwann cells can occur in the absence of beta4 integrin demonstrating that this Schwann cell component is dispensable for myelin formation in peripheral nerves.

Loss of distal axons and sensory Merkel cells and features indicative of muscle denervation in hindlimbs of P0-deficient mice.

Mice lacking the major Schwann cell myelin component P0 show a severe dysmyelination with pathological features reminiscent of the Déjérine-Sottas syndrome in humans. Previous morphological and electrophysiological studies on these mice did not only demonstrate a compromised myelination and myelin maintenance, but were suggestive of an impairment of axons as well. Here, we studied the axonal pathology in P0-deficient mice by quantitative electron microscopy. In addition, we investigated epidermal receptor end organs by immunocytochemistry and muscle pathology by histochemistry. In proximal sections of facial and femoral nerves, axon calibers were significantly reduced, whereas the number of myelin-competent axons was not diminished in 5- and 17-month-old P0-deficient mice. However, in distal branches of the femoral and sciatic nerve (digital nerves innervating the skin of the first toe) the numbers of myelin-competent axons were reduced by 70% in 6-month-old P0-deficient mice. Immunolabeling of foot pads revealed a corresponding loss of Merkel cells by 75%, suggesting that survival of these cells is dependent on the presence or maintenance of their innervating myelinated axons. In addition, quadriceps and gastrocnemius muscles showed pathological features indicative of denervation and axonal sprouting. These findings demonstrate that loss of an important myelin component can initiate degenerative mechanisms not only in the Schwann cell but also in the distal portions of myelinated axons, leading to the degeneration of specialized receptor end organs and impairment of muscle innervation.

Motor neuropathy in porphobilinogen deaminase-deficient mice imitates the peripheral neuropathy of human acute porphyria.

Acute porphyrias are inherited disorders caused by partial deficiency of specific heme biosynthesis enzymes. Clinically, porphyrias are manifested by a neuropsychiatric syndrome that includes peripheral neuropathy. Although much is known about the porphyrias' enzyme defects and their biochemical consequences, the cause of the neurological manifestations remains unresolved. We have studied porphyric neuropathy in mice with a partial deficiency of porphobilinogen deaminase (PBGD). PBGD-deficient mice (PBGD-/-) imitate acute porphyria through massive induction of hepatic delta-aminolevulinic acid synthase by drugs such as phenobarbital. Here we show that PBGD-/- mice develop impairment of motor coordination and muscle weakness. Histologically femoral nerves of PBGD-/- mice exhibit a marked decrease in large-caliber (>8 microm) axons and ultrastructural changes consistent with primary motor axon degeneration, secondary Schwann cell reactions, and axonal regeneration. These findings resemble those found in studies of affected nerves of patients with acute porphyria and thus provide strong evidence that PBGD deficiency causes degeneration of motor axons without signs of primary demyelination, thereby resolving a long-standing controversy. Interestingly, the neuropathy in PBGD-/- mice developed chronically and progressively and in the presence of normal or only slightly (twofold) increased plasma and urinary levels of the putative neurotoxic heme precursor delta-aminolevulinic acid. These data suggest that heme deficiency and consequent dysfunction of hemeproteins can cause porphyric neuropathy.

The impact of a muscle target organ on nerve grafts with different lengths--a histomorphological analysis.

The present study was done in order to evaluate the influence of a target muscle on the regenerative processes in long nerve grafts. In 21 rabbits the saphenous nerve was used as a nerve graft and coapted to the cut motor nerve of vastus medialis. The animals were separated into three groups with different graft lengths, namely 3, 5, and 7 cm. In a second stage the distal end of the graft (Graft.dist.) was coapted to the motor branch of rectus femoris. Cross sections of the normal vastus nerve and the Graft.dist. before and 7 months after the connection to rectus femoris were analyzed histomorphometrically. Before coaptation to the target organ mean fiber number in the Graft.dist. of the 3-cm-long grafts was 3380 and decreased to 2413 in the 7-cm-long grafts. Seven months after coaptation the results showed a statistically significant decrease of fibers in the Graft.dist. of group two and three and a distinct decrease of the fibers in group one. Summarizing, in a two-stage nerve grafting procedure the reinnervation of the muscle target organ leads to a down-regulation of fibers in the distal end of short and long nerve grafts.

Ultrastructural study of peripheral nerve injury induced by monopolar and bipolar diathermy.

The effect of unipolar and bipolar electrocoagulation on human and albino rat peripheral nerves was examined by transmission electron microscopy. The greater auricular nerve obtained from operated patients and the femoral nerve of an albino rat were chosen for this study. The nerves were divided into three groups: controls, segments of nerves spot-coagulated with unipolar instruments, and segments of nerves spot-coagulated with bipolar instruments. Significant cell injury, expressed by marked damage of the Schwann cells, was induced by unipolar coagulation in both myelinated and unmyelinated nerves, in humans as well as in the albino rat. However, in bipolar-treated nerves, the Schwann cell architecture remained well preserved in both types of nerves. The results are in favor of bipolar electrocoagulation when applied in areas rich in neurovascular supplies.

Crucial role for the myelin-associated glycoprotein in the maintenance of axon-myelin integrity.

It has recently been shown that mice deficient in the gene for myelin-associated glycoprotein develop normal myelin sheaths in the peripheral nervous system. Here we report that in mutant mice older than 8 months the maintenance of axon-myelin units is disturbed, resulting in both axon and myelin degeneration. Morphological features include those typically seen in human peripheral neuropathies, where demyelination-induced Schwann cell proliferation and remyelination lead to the formation of so-called onion bulbs. Expression of tenascin-C, a molecule indicative of peripheral nerve degeneration, was up-regulated by axon-deprived Schwann cells and regenerating axons were occasionally seen. Myelin-associated glycoprotein thus appears to play a crucial role in the long-term maintenance of the integrity of both myelin and axons.

Changes in the myelinated axons of femoral nerve in amyotrophic lateral sclerosis.

Myelinated fibres in femoral nerves removed from amyotrophic lateral sclerosis (ALS) cases at post mortem were compared with age matched controls. A technique for processing whole transverse sections of the nerves for osmication and subsequent morphometric analysis is described. Although areas depleted in myelinated fibres were seen in the nerves from the ALS group, no statistically significant difference was shown due to wide variations in the controls. However, the ALS nerves showed a degree of disruption in the myelin which was not apparent in the controls. The most obvious effect was widespread "wrinkling" of the myelin in both large and small fibres from the ALS nerves. This phenomenon is the initial stage of a process which eventually results in uneven myelin thickness and nodal swellings and finally myelin ovoids and balls. We illustrate the steps in the progression of this degeneration with teased nerve studies and electron microscopy and propose that there are qualitative changes in the myelin of peripheral nerve in ALS. It seems likely that these are secondary effects resulting from axonal degeneration caused by deterioration and loss of anterior horn cells in the spinal cord.