Effect of age and muscle type on regeneration of neuromuscular synapses in mice

A silver stain was used to investigate the regeneration of nerve terminals in mouse diaphragm, superior gluteus and tensor fasciae latae (TFL) muscles following nerve crush injury at different ages (9-530 days). The development of myelinated terminal branches and the development and elimination of terminal sprouts were little affected by age or muscle type following reinnervation. However, multiple axonal innervation developed on up to 50% of the gluteus and TFL muscle fibres, and this was subsequently eliminated only in the youngest animals.     

Effects of advancing age on peripheral nerve regeneration.

Following axotomy, the regrowth of peripheral axons takes longer in older individuals than in young ones. The present study compares the crush-induced process of degeneration and regeneration in the buccal branch of the facial motor nerve in groups of rats aged 3 months and 15 months. Observations are based on qualitative and quantitative analyses of the nerve 20 mm from the site of injury in rats 1, 2, 4, 16, 21, 28, and 56 days after crush. The buccal branch is purely motor and contains a unimodal population of about 1,600 axons commonly in a single fascicle. During the first 28 days post crush (dpc) in the 3-month animals, the progression of myelin and axon degeneration, myelin clearance, regrowth of axon sprouts, and axon maturation are relatively synchronized and uniform. In the older rats, the degeneration of myelin and axons, myelin clearance, and the appearance of axon sprouts at the site of sample are all delayed. In the younger animals, axon sprouts increase in numbers from their first appearance at 4 dpc through the 2 weeks examined following the restoration of whisking behavior. The numbers of regenerating older axons increase at a rate comparable to that in the younger animals through the time that bilaterally symmetrical whisking behavior is evident, but afterwards the number of axon sprouts decreases. At 2 months after crush the young animals have 30% more fibers in the buccal branch than control nerves, while the older animals have fewer than control numbers. In the 3-month regenerated nerve, 2 months post crush, 30% of the regenerated fibers are of very small caliber, less than 3 microns2 in cross sectional area, and typically these small axons have unusually thick myelin sheaths; the older nerves do not have such a skewed distribution of axon areas. The older regenerated axons at 2 months post crush have an unusually high density of microtubules compared to the younger regenerated ones (and controls), and the ratio of neurofilaments to microtubules is very low. The conclusions are that motor neurons in older animals regenerate damaged axons after a delay not apparent in the young; the strong regenerative response apparent initially in animals of both age groups is not maintained in the older animals; and the relationship between the numerical density of cytoskeletal elements and the axon cross-sectional area deviates from normal in the regenerated axons of the older animals.     

Delayed olfactory nerve regeneration in ApoE-deficient mice

Apolipoprotein E (apoE), a lipid transporting protein, is extensively expressed in the primary olfactory pathway, but its function is unknown. We previously reported increased apoE levels in the olfactory bulb (OB) following olfactory epithelium (OE) lesion in mice, and hypothesized that apoE may play a vital role in olfactory nerve (ON) regeneration. To directly test this hypothesis, we examined the rate of ON regeneration following OE lesion in apoE deficient/knockout (KO) and wild-type (WT) mice. OE was lesioned in 2- to 3-month-old mice by intranasal irrigation with Triton X-100 (TX). OB were collected at 0, 3, 7, 21, 42, and 56 days post-lesion. OB recovery was measured by both immunoblotting and immunohistochemical analysis of growth cone associated protein (GAP) 43 and olfactory marker protein (OMP). The results revealed that (1) OMP recovery in the OB was significantly slower in apoE KO compared to WT mice; (2) recovery of glomerular area was similarly slower; and (3) GAP43 increases and return to prelesion levels in the OB were slower in KO mice. Together, these results show that olfactory nerve regeneration is significantly slower in KO mice as compared to WT mice, suggesting apoE facilitates olfactory nerve regeneration.     

Impaired nerve regeneration in reeler mice after peripheral nerve injury

Reelin, an extracellular matrix protein, plays an important role in the regulation of neuronal migration and cortical lamination in the developing brain. Little is known, however, about the role of this protein in axonal regeneration. We have previously shown that Reelin is secreted by Schwann cells in the peripheral nerve compartment during postnatal development and that it is up-regulated following nerve injury in adult mice. In this work, we generated mice deficient in Reelin ( reeler ) that express yellow fluorescent protein (YFP) in a subset of neurons and examined the axonal regeneration following nerve crush. We found that axonal regeneration was significantly altered compared with wild-type mice. By contrast, retrograde tracing with Fluorogold dye after sciatic nerve crush was unaffected in these mutants, being comparable with normal axonal transport observed in wild-type. These results indicate that the absence of Reelin impairs axonal regeneration following injury and support a role for this protein in the process of peripheral nerve regeneration.     

The effect of a conditioning lesion on sudomotor axon regeneration

The effects of a conditioning lesion on the rate of sudomotor axon regeneration were judged by the recovery of sweat gland (SG) secretion after cholinergic stimulation. Three groups of mice were given a conditioning lesion by crushing the sciatic nerve at mid-thigh 4, 7, and 14 days before a test lesion. A 4th group received a conditioning crush of the tibial nerve at the ankle 7 days before the test lesion. Control mice had a single test lesion. SG reinnervation in control mice began 19 days after the test lesion, and was functionally complete by 41 days. In groups with the conditioning lesion 4, 7, and 14 days before the test operation, the first reactive SGs reappeared at 16, 15, and 16 days respectively after the test lesion, and maximal recovery occurred by 33, 32, and 39 days. In mice with the distal conditioning lesion, reinnervation began at 19 days and was maximal by 36 days. In summary, a nerve conditioning lesion placed from 4 to 14 days prior to and at the same site as a test lesion significantly accelerated the growth rate of the fastest regenerating unmyelinated sudomotor axons and reduced the time until most SGs were reinnervated. A more distally placed test lesion reduced the interval for recovery.     

The effects of age and ganglioside composition on the rate of motor nerve terminal regeneration following antibody-mediated injury in mice

Gangliosides are glycosphingolipids highly enriched in neural plasma membranes, where they mediate a diverse range of functions and can act as targets for auto-antibodies present in human immune-mediated neuropathy sera. The ensuing autoimmune injury results in axonal and motor nerve terminal (mNT) degeneration. Both aging and ganglioside-deficiency have been linked to impaired axonal regeneration. To assess the effects of age and ganglioside expression on mNT regeneration in an autoimmune injury paradigm, anti-ganglioside antibodies and complement were applied to young adult and aged mice wildtype (WT) mice, mice deficient in either b- and c-series (GD3sKO) or mice deficient in all complex gangliosides (GM2sKO). The extent of mNT injury and regeneration was assessed immediately or after 5 days, respectively. Depending on ganglioside expression and antibody-specificity, either a selective mNT injury or a combined injury of mNTs and neuromuscular glial cells was elicited. Immediately after induction of the injury, between 1.5% and 11.8% of neuromuscular junctions (NMJs) in the young adult groups exhibited healthy mNTs. Five days later, most NMJs, regardless of age and strain, had recovered their mNTs. No significant differences could be observed between young and aged WT and GM2sKO mice; aged GD3sKO showed a mildly impaired rate of mNT regeneration when compared with their younger counterparts. Comparable rates were observed between all strains in the young and the aged mice. In summary, the rate of mNT regeneration following anti-ganglioside antibody and complement-mediated injury does not differ majorly between young adult and aged mice irrespective of the expression of particular gangliosides. Synapse 67:382–389, 2013. © 2013 Wiley Periodicals, Inc.     

Effect of age and gender on sudomotor and cardiovagal function and blood pressure response to tilt in normal subjects

Normative data are limited on autonomic function tests, especially beyond age 60 years. We therefore evaluated these tests in a total of 557 normal subjects evenly distributed by age and gender from 10 to 83 years. Heart rate (HR) response to deep breathing fell with increasing age. Valsalva ratio varied with both age and gender. QSART (quantitative sudomotor axon-reflex test) volume was consistently greater in men (approximately double) and progressively declined with age for all three lower extremity sites but not the forearm site. Orthostatic blood pressure reduction was greater with increasing age. HR at rest was significantly higher in women, and the increment with head-up tilt fell with increasing age. For no tests did we find a regression to zero, and some tests seem to level off with increasing age, indicating that diagnosis of autonomic failure was possible to over 80 years of age. © 1997 John Wiley & Sons, Inc. Muscle Nerve 20: 1561–1568, 1997     

17Beta-estradiol stimulates regeneration of sciatic nerve in female mice

In ovariectomized mice with and without estrogen replacement, regeneration of the sciatic nerve after crush injury was studied. Functional recovery, quantified with sciatic functional index was significantly accelerated in estrogen-treated mice throughout the regeneration. On semi-thin sections of sciatic nerves in estrogen-treated mice we registered a greater total number of regenerating nerve fibers at the first week, and a higher mean axonal area at the third week of regeneration. Our results demonstrated that estrogen treatment enhances regeneration of the sciatic nerve.     

Muscle coordination in hindlimbs of mice after bilateral nerve regeneration

The common peroneal nerve was transected bilaterally in 25 adult mice. Nerve stumps were immediately readapted without nerve suture. Before transection and after nerve regeneration the muscle coordination of tibialis anterior (TA) and medial gastrocnemius (MG) muscle was examined by electromyographic recordings from both muscles (EMG) during free running. Using a personal computer, the degree of muscle coordination between TA and MG was determined by calculating a coordination index. In normal mice an antagonistic innervation pattern was observed. After nerve transection and regeneration the degree of muscle coordination of TA and MG substantially decreased with great interindividual but also great intraindividual variation. In 16 mice there was no correlation between the coordination index of the left and right hindlimbs. In nine out of 25 mice reinnervation was absent on one side. These results suggest that nerve regeneration by axonal sprouting to appropriate or foreign muscles occurs at random and that there are no intraindividual factors which might promote the finding of the proper target muscle.     

Changes in sudomotor nerve territories with aging in the mouse

This study evaluates sudomotor function in the hindpaw of young and aged mice. Sweating was stimulated by pilocarpine injection and by electrical stimulation of the sciatic, tibial, peroneal, sural and saphenous nerves. The number of responsive sweat glands of the paw was determined by the silicon mold technique. The results obtained provide evidence that the number of functioning sweat glands of the hindpaw tended to decrease in aged mice. The peripheral sudomotor territories and the complement of sweat glands for individual nerves declined slightly with age. Moreover, the number of sweat glands responsive to cholinergic stimulation was decreased when compared with the number responsive to electrical stimulation of the sciatic nerve in aged mice. These and previous observations suggest that the number of sudomotor axons in the peripheral nerve, as well as their capabilities for compensatory reinnervation of sweat glands by regeneration and by sprouting, is reduced with aging.     

Magnetic stimulation supports muscle and nerve regeneration after trauma in mice

Introduction: Magnetic stimulation (MS) has the ability to induce muscle twitch and has long been proposed as a therapeutic modality for skeletal muscle diseases. However, the molecular mechanisms underlying its means of action have not been defined. Methods: Muscle regeneration after trauma was studied in a standard muscle injury mouse model. The influence of MS on the formation of motor units, posttrauma muscle/nerve regeneration, and vascularization was investigated. Results: We found that MS does not cause systemic or muscle damage but improves muscle regeneration by significantly minimizing the presence of inflammatory infiltrate and formation of scars after trauma. It avoids posttrauma muscle atrophy, induces muscle hypertrophy, and increases the metabolism and turnover of muscle. It triples the expression of muscle markers and significantly improves muscle functional recovery after trauma. Conclusions: Our results indicate that MS supports muscle and nerve regeneration by activating muscle–nerve cross‐talk and inducing the maturation of neuromuscular junctions. Muscle Nerve 53 : 598–607, 2016     

Glia maturation factor beta stimulates axon regeneration in transected rat sciatic nerve.

Rat sciatic nerves were bilaterally transected and repaired with an entubulation technique. The nerve interstump gap was filled with either collagen gel or collagen gel mixed with a putative neurotrophic factor (leupeptin, 4-aminopyridine, lipid angiogenic factor or glia maturation factor beta (GMF-beta]. Six weeks after nerve transection, the myelinated distal stump axons were quantified for each nerve. Only the nerves treated with GMF-beta had significantly more axons than the control side.     

Inhibitor of DNA binding 2 accelerates nerve regeneration after sciatic nerve injury in mice

Inhibitor of DNA binding 2 (Id2) can promote axonal regeneration after injury of the central nervous system. However, whether Id2 can promote axonal regeneration and functional recovery after peripheral nerve injury is currently unknown. In this study, we established a mouse model of bilateral sciatic nerve crush injury. Two weeks before injury, AAV9-Id2-3×Flag-GFP was injected stereotaxically into the bilateral ventral horn of lumbar spinal cord. Our results showed that Id2 was successfully delivered into spinal cord motor neurons projecting to the sciatic nerve, and the number of regenerated motor axons in the sciatic nerve distal to the crush site was increased at 2 weeks after injury, arriving at the tibial nerve and reinnervating a few endplates in the gastrocnemius muscle. By 1 month after injury, extensive neuromuscular reinnervation occurred. In addition, the amplitude of compound muscle action potentials of the gastrocnemius muscle was markedly recovered, and their latency was shortened. These findings suggest that Id2 can accelerate axonal regeneration, promote neuromuscular reinnervation, and enhance functional improvement following sciatic nerve injury. Therefore, elevating the level of Id2 in adult neurons may present a promising strategy for peripheral nerve repair following injury. The study was approved by the Experimental Animal Ethics Committee of Jinan University (approval No. 20160302003) on March 2, 2016.

Chinese Library Classification No. R456; R745; R364.3+3     

Effects of tacrolimus （FK506） on sciatic nerve regeneration in rats

BACKGROUND： Tacrolimus （FK506） protects peripheral nerves located in damaged regions by inhibiting T lymphocyte proliferation and activation. OBJECTIVE： To evaluate the effect of FK506 on promoting regeneration of rat sciatic nerve. DESIGN, TIME AND SETTING： A randomized, controlled, animal study was performed at the Laboratory of the Department of Orthopedic Surgery, Dalian Medical University, China, from September 2007 to September 2008. MATERIALS： A total of 60 adult, male, Sprague-Dawley rats were equally and randomly divided into model, local administration and systemic administration groups. All rats received a neurotomy of bilateral sciatic nerves to establish models of nerve regeneration chambers. The powder and injection of FK506 were supplied by Fujisawa Pharmaceutical, Japan. METHODS： The regeneration chambers of the model group were infused with 0.2 mL saline. The systemic group were injected with 0.2 mL saline, followed by daily subcutaneous injections of FK506 （1 mg/kg）, for 14 days. The local administration group was infused with 0.2 mL FK506 （1 μg/mL）. MAIN OUTCOME MEASURES： Local immune response was observed using hematoxylin-eosin staining. Myelinated nerve fiber number, myelin sheath and nerve fiber thickness were observed using toluidine blue staining. Wet weight of gastrocnemius was evaluated. Compound muscle action potential amplitude, latency, and conduction time were recorded, and motor nerve conduction velocity was calculated using electrophysiology. RESULTS： The total number of myelinated nerve fibers in the local and systemic administration groups was significantly higher than in the model group. The density of myelinated nerve fibers, myelin sheath thickness and mean axon diameter were significantly increased in the systemic administration group compared with the model group （P 〈 0.05）. Lymphocyte infiltration was decreased in the local and systemic administration groups compared with the model group. The wet weight of rat gastrocnemius in the local and systemic administration groups were significantly greater compared with the model group （P 〈 0.05）. Motor nerve conduction velocity was the fastest in the systemic administration group, and the slowest in the model group. Compound muscle action potential amplitude was larger in the systemic administration group compared with the local administration and model groups （P 〈 0.05）. CONCLUSION： Systemic administration of FK506 can promote regeneration of rat sciatic nerve and recovery of neural function. Systemic administration produced better regeneration and recovery of function than local administration of FK506.     

Polymer electret guidance channels enhance peripheral nerve regeneration in mice

Polytetrafluoroethylene (PTFE) tubes were prepared as electrets displaying a quasi-permanent surface charge due to the presence of trapped monopolar charge carriers. PTFE tubes containing either positive or negative charges and electrically neutral PTFE tubes were used as nerve guidance channels for the repair of a ⁴mm nerve gap in the sciatic nerve of mice. After 4 weeks of implantation, positively and negatively charged PTFE electrets contained regenerated nerves with significantly more myelinated axons than nerves regenerated in uncharged PTFE tubes. This observation suggests that peripheral nerve regeneration can be enhanced by electrically charged nerve guidance channels.     

Sciatic nerve regeneration is accelerated in galectin-3 knockout mice

The success of peripheral nerve regeneration depends on intrinsic properties of neurons and a favorable environment, although the mechanisms underlying the molecular events during degeneration and regeneration are still not elucidated. Schwann cells are considered one of the best candidates to be closely involved in the success of peripheral nerve regeneration. These cells and invading macrophages are responsible for clearing myelin and axon debris, creating an appropriate route for a successful regeneration. After injury, Schwann cells express galectin-3, and this has been correlated with phagocytosis; also, in the presence of galectin-3, there is inhibition of Schwann-cell proliferation in vitro. In the present study we explored, in vivo, the effects of the absence of galectin-3 on Wallerian degeneration and nerve-fiber regeneration. We crushed the sciatic nerves of galectin-3 knockout and wild-type mice, and followed the pattern of degeneration and regeneration from 24 h up to 3 weeks. We analyzed the number of myelinated fibers, axon area, fiber area, myelin area, G-ratio and immunofluorescence for β-catenin, macrophages and Schwann cells in DAPI counterstained sections. Galectin-3 knockout mice showed earlier functional recovery and faster regeneration than the wild-type animals. We concluded that the absence of galectin-3 allowed faster regeneration, which may be associated with increased growth of Schwann cells and expression of β-catenin. This would favor neuron survival, followed by faster myelination, culminating in a better morphological and functional outcome.     

Peripheral nerve regeneration is delayed in neuropilin 2-deficient mice

Peripheral nerve transection or crush induces expression of class 3 semaphorins by epineurial and perineurial cells at the injury site and of the neuropilins neuropilin-1 and neuropilin-2 by Schwann and perineurial cells in the nerve segment distal to the injury. Neuropilin-dependent class 3 semaphorin signaling guides axons during neural development, but the significance of this signaling system for regeneration of adult peripheral nerves is not known. To test the hypothesis that neuropilin-2 facilitates peripheral-nerve axonal regeneration, we crushed sciatic nerves of adult neuropilin-2-deficient and littermate control mice. Axonal regeneration through the crush site and into the distal nerve segment, repression by the regenerating axons of Schwann cell p75 neurotrophin receptor expression, remyelination of the regenerating axons, and recovery of normal gait were all significantly slower in the neuropilin-2-deficient mice than in the control mice. Thus, neuropilin-2 facilitates peripheral-nerve axonal regeneration.     

Effect of a conditioning lesion on optic nerve regeneration in goldfish

Following a ‘testing lesion’ (crush) of the optic nerve in goldfish, histological study of axons in silver-stained sections showed that outgrowth of the leading axons began after an initial delay of 4.3 days and proceeded at0.34 ± 0.03mm/day. When a ‘conditioning lesion’ (crush at the same site) preceded the testing lesion by 2 weeks, the initial delay was 2.5 days and the outgrowth rate was0.74 ± 0.13mm/day (P < 0.01).Two additional methods, utilizing intraocular injections of tritiated proline or fucose to label axonally transported proteins, were used to examine the outgrowth of leading optic axons. (a) Measurement of the distances reached by labeled axons in the nerve at 6 and 10 days after a testing lesion alone yielded an initial delay of 4.6 days and an outgrowth rate of0.41 ± 0.04mm/day. However, when a conditioning lesion preceded the testing lesion, labeled optic axons were already found to have reached the optic tectum by 10 days after the testing lesion, indicating an outgrowth rate in excess of 0.64 mm/day. (b) Determination of the times at which labeled axons arrived at the optic tectum showed that the outgrowth rate after a testing lesion alone was 0.40 mm/day whereas when the testing lesion was preceded by a conditioning lesion it was 0.74 mm/day.Thus, as a result of a conditioning lesion the initial delay was reduced bynearly half and the outgrowth rate was nearly doubled.     

神经生长因子促再生坐骨神经血管生成的作用

BACKGROUND： It remains to be determined whether nerve growth factor （NGF） can promote angiogenesis in regenerating peripheral nerves during repairing peripheral nerve injury.
OBJECTIVE： To evaluate the effects of NGF on angiogenesis, and to analyze the influencing mechanisms of NGF, according to the expression patterns of CD34, von Willebrand factor （vWF）, vascular endothelial cell growth factor （VEGF）, and the NGF receptor TrkA in proliferating vascular endothelial cells from a rat model of sciatic nerve injury.
DESIGN, TIME AND SETTING： Randomized, controlled study performed at the Research Institute of Field Surgery, Daping Hospital affiliated to the Third Military Medical University of Chinese PLA, between October 2003 and July 2005.
MATERIALS： Forty-five healthy, adult, Wistar rats underwent sciatic nerve injury. The rats were randomly divided into four groups： NGF ＋ chitosan （n = 15）, NGF ＋ chitosan ＋ anti-VEGF （n = 10）, chitosan （n = 10）, and physiological saline （n = 10）. METHODS： A 1 -cm defected sciatic nerve was bridged with a silica gel conduit. NGF ＋ chitosan group： 100 μ L chitosan and 5 μ L NGF （20 mg/L） were injected into the silica gel conduit; NGF ＋ chitosan ＋ anti-VEGF group： an additional 5μ L anti-VEGF monoclonal antibody （1 g/L） was injected into the silica gel conduit; chitosan group： 100μL chitosan and 5 μL physiological saline were injected into the silica gel conduit; physiological saline group： only 5μL physiological saline was injected into the silica gel conduit.
MAIN OUTCOME MEASURES： CD34 and vWf were used to label blood capillaries and large-diameter blood vessels in the regenerating peripheral nerves, respectively. At day 14 following surgery, immunohistochemistry was used to detect and semi-quantitatively analyze expressions of CD34, vWf, VEGF, and TrkA in proliferating vascular endothelial cells in the regenerating sciatic nerve. A confocal laser microscope was used to determine co-expression. RESULTS： Expressions of TrkA, CD34, vWf, and VEGF in the NGF ＋ chitosan group were significantly greater than the physiological saline and chitosan groups （P 〈 0.05-0.01）. Expressions of CD34 and VEGF in the NGF ＋ chitosan ＋ anti-VEGF group were completely inhibited, while expressions of vWf and TrkA gradually decreased, compared with the NGF ＋ chitosan group （P 〈 0.01）. Confocal microscopy revealed strong co-expression of VEGF and CD34 in the regenerating sciatic nerve, and CD34 expression positively correlated with VEGF expression. In addition, VEGF expression was greater than CD34 expression, and coexpression of VEGF and vWf was also strong.
CONCLUSION： VEGF was expressed in blood capillaries and large-diameter blood vessels, while exogenous NGF promoted VEGF expression in regenerating sciatic nerves, thereby increasing angiogenesis.