Density Of Myelinated Nerve Fibers Research Articles

Influence of shortwave laser therapy on morphometric parameters of sciatic nerve regeneration after experimental neurorrhaphy

The aim of this work was to reveal the effectiveness of delayed laser therapy for the regeneration of the sciatic nerve. Materials and methods. In 21 rats, transection and epineural microsurgical suture of the right sciatic nerve (SN) were performed. In 16 rats (experimental group) in the period from 1.0 to 2.5 months after surgery, a course of laser therapy (LT-1, n = 8) and a delayed (after 6 months) course of laser therapy (LT-2, n = 8) were used 18 sessions were performed in each subgroup. Laser therapy was not performed in 5 operated animals and they were included in the control group. The terms of withdrawal of rats from the experiment were 2.5 and 12.0 months after the operation. During the morphometry of semi-thin sections distal to the level of the sutures, the volumetric density of the constituent elements of the SN, the number density of myelinated nerve fibers (MNF), their diameters, the thickness of the axons and myelin sheath, the myelination index (index G) distribution of MNF by diameter and dependence of index G on the fiber caliber were measured. For double control, we used muscle morphometry data from intact rats (Intact). Muscle tissue was additionally examined by the method of transmission electron microscopy using a generally accepted technique with a REM-100 electron microscope (Selmi, Sumy, Ukraine). Results. In the control group, as compared to the intact group, the numerical density of muscle fibers was 21 % higher, but 38 % less than the numerical density of microvessels, therefore, the vascularization index was reduced by 1.7 times. The obtained results indicated laser compensation for denervation atrophy, which was confirmed by morphometric data. Conclusions. The results of the study indicate a stimulating prolonged effect of delayed laser therapy in regenerative axonogenesis.

Quantification of heat shock proteins in the posterior interosseous nerve among subjects with type 1 and type 2 diabetes compared to healthy controls.

Diabetic peripheral neuropathy (DPN) is a common complication of both type 1 (T1D) and type 2 diabetes (T2D). No cure for DPN is available, but several potential targets have been proposed for treatment. Heat shock proteins (HSPs) are known to respond to both hyper- and hypoglycemia. DPN can be diagnosed using electrophysiology and studied using peripheral nerve biopsies. This study aimed to analyze the presence and patterns of HSPs in peripheral nerve biopsies from subjects with T1D, T2D, and healthy controls. Posterior interosseous nerves (PIN) from a total of 56 subjects with T1D (n = 9), with T2D (n = 24), and without diabetes (i.e., healthy controls, n = 23) were harvested under local anesthesia and prepared for quantitative mass spectrometry analysis. Protein intensities were associated with electrophysiology data of the ulnar nerve and morphometry of the same PIN, and differences in protein intensities between groups were analyzed. In total, 32 different HSPs were identified and quantified in the nerve specimens. No statistically significant differences were observed regarding protein intensities between groups. Furthermore, protein intensities did not correlate with amplitude or conduction velocity in the ulnar nerve or with the myelinated nerve fiber density of PIN. Quantitative proteomics can be used to study HSPs in nerve biopsies, but no clear differences in protein quantities were observed between groups in this cohort.

322-OR: Proinflammatory Macrophage Infiltration in Sural Nerve Is Associated with Reduction of Myelinated Nerve Fiber Density but Not IENFD in Human Diabetic Subjects

Background: The etiology of diabetic polyneuropathy (DPN) is multifactorial, and other than hyperglycemia, circulatory disturbance such as hypertension can be involved. Pro-inflammatory macrophages (M1) infiltration in the peripheral nerve is known to be the pathogenesis of DPN. However, it is still unclear how the inflammation and circulatory disturbance are involved in the pathological changes of human DPN. Methods: Autopsied cases at Hirosaki University Hospital were evaluated: 7 nondiabetic cases (nDM), 11 nondiabetic cases with hypertension (nDM+HT), 6 diabetes cases (DM), 9 cases with hypertension and diabetes (DM+HT). Intraepidermal nerve fiber density (IENFD) was examined by immunofluorescent staining with the skin tissue 10 cm above external condyle. Dissected sural nerves were pathologically examined. Macrophage infiltration into the endoneurium of sural nerves was evaluated by double immunostaining using anti-CD68 and anti-CD163 antibodies. Results: HbA1c level was significantly higher in DM and DM+HT than in nDM (p<0.01, respectively). IENFD was significantly decreased in DM compared to nDM (p<0.05) and further decreased in DM+HT (p<0.05 vs DM). The infiltration of M1 was significantly increased in DM (p<0.05). M1 infiltration was comparable between DM and DM+HT. Regression analysis showed that the M1/anti-inflammatory macrophages (M2) ratio was not significantly correlated with IENFD (r=0.3, p=0.09). Morphometric measurements of the sural nerve using semithin sections revealed that myelinated nerve fiber density (MNFD) was significantly decreased in DM compared with nDM (p<0.05). MNFD was comparable between DM and DM+HT. MNFD was inversely correlated with M1/M2 ratio (r=0.37, p<0.05). Conclusions: Our results suggest that the factors other than sural nerve inflammation, such as hypertension, are possibly more involved in the loss of IENFD in DPN. Disclosure H. Mizukami: None. S. Osonoi: None. Y. Takeuchi: None. T. Sasaki: None.

CIDP/autoimmune nodopathies with nephropathy: a case series study.

The co-morbidity of chronic inflammatory demyelinating polyradiculoneuropathy (CIDP)/autoimmune nodopathies with nephropathy has been gradually known in recent years. This study was intended to explore the clinical, serological and neuropathological features of seven patients with CIDP/autoimmune nodopathies and nephropathy. Among 83 CIDP patients, seven were identified with nephropathy. Their clinical, electrophysiological and laboratory examination data were collected. The nodal/paranodal antibodies were tested. The sural biopsies were performed in all the patients, and renal biopsies were operated in 6 patients. Six patients had chronic onsets and one had an acute onset. Four patients exhibited peripheral neuropathy preceding nephropathy while two showed concurrent onset of neuropathy and nephropathy, and one started with nephropathy. All the patients showed demyelination in electrophysiological examination. Nerve biopsies showed mild to moderate mixed neuropathies including demyelinating and axonal changes in all patients. Renal biopsies showed membranous nephropathy in all 6 patients. Immunotherapy was effective in all patients, with two patients showing good response to corticosteroid treatment alone. Four of the patients were positive to anti-CNTN1 antibody. Compared with anti-CNTN1 antibody-negative patients, antibody-positive patients had a higher proportion of ataxia (3/4 vs. 1/3), autonomic dysfunction (3/4 vs. 1/3), less frequent antecedent infections (1/4 vs. 2/3), higher cerebrospinal fluid proteins (3.2g/L vs. 1.69 g/L), more frequent conduction block on electrophysiological examination (3/4 vs. 1/3), higher myelinated nerve fiber density, and positive CNTN1 expression in the glomeruli of kidney tissues. Anti-CNTN1 antibody was the most frequent antibody in this group of patients with CIDP/autoimmune nodopathies and nephropathy. Our study suggested that there might be some clinical and pathological differences between the antibody positive and negative patients.

Comparative analysis of intracardiac neural structures in the aged rats with essential hypertension.

Persistent arterial hypertension initiates cardiac autonomic imbalance and alters cardiac tissues. Previous studies have shown that neural component contributes to arterial hypertension etiology, maintenance, and progression and leads to brain damage, peripheral neuropathy, and remodeling of intrinsic cardiac neural plexus. Recently, significant structural changes of the intracardiac neural plexus were demonstrated in young prehypertensive and adult hypertensive spontaneously hypertensive rats (SHR), yet structural alterations of intracardiac neural plexus that occur in the aged SHR remain undetermined. Thus, we analyzed the impact of uncontrolled arterial hypertension in old (48-52 weeks) SHR and the age-matched Wistar-Kyoto rats (WKY). Intrinsic cardiac neural plexus was examined using a combination of immunofluorescence confocal microscopy and transmission electron microscopy in cardiac sections and whole-mount preparations. Our findings demonstrate that structural changes of intrinsic cardiac neural plexus caused by arterial hypertension are heterogeneous and may support recent physiological implications about cardiac denervation occurring together with the hyperinnervation of the SHR heart. We conclude that arterial hypertension leads to (i) the decrease of the neuronal body area, the thickness of atrial nerves, the number of myelinated nerve fibers, unmyelinated axon area and cumulative axon area in the nerve, and the density of myocardial nerve fibers, and (ii) the increase in myelinated nerve fiber area and density of neuronal bodies within epicardiac ganglia. Despite neuropathic alterations of myelinated fibers were exposed within intracardiac nerves of both groups, SHR and WKY, we consider that the determined significant changes in structure of intrinsic cardiac neural plexus were predisposed by arterial hypertension.

Clinical feature and sural biopsy study in nitrous oxide-induced peripheral neuropathy.

ObjectiveThe objective was to analyze the clinical characteristics and pathological characteristics of sural biopsy in nitrous oxide (N2O) -induced peripheral neuropathy.MethodsWe recruited 18 patients with N2O abuse-induced neurological disorders and reported their demographic data, clinical manifestations, laboratory examinations, and nerve conduction studies. Seven patients underwent sural nerve biopsy pathologic examination.ResultsAll 18 patients had polyneuropathy, the nerve conduction results showed significant reductions in motor and sensory amplitudes, slowing of conduction velocities, and prolongation of latencies in most tested nerves compared to the controls. Toluidine blue staining of semi-thin sections of sural nerve biopsy showed decreased myelinated nerve fiber density, increased thin myelinated nerve fiber density, and axonal regeneration. Electron microscopy showed axonal degeneration and nerve regeneration.ConclusionThe main manifestations of peripheral nerve damage caused by the abuse of N2O are lower limb weakness and distal sensory disorder. The nerve conduction study results demonstrated that mixed axonal and demyelinating neuropathy was the most common type of neuropathy. Sural biopsy showed the main pathological change was chronic axonal degeneration.

Comparison of the vitality tests used in the dental clinical practice and histological analysis of the dental pulp.

In dentistry, indirect diagnostic methods such as electrical sensibility testing and pulse oximetry are used to assess the status of the pulp. Our study aimed to determine the correlation between hemoglobin oxygen saturation and vascular volume density (Vvasc). We also wanted to examine an electrical sensibility test and the volume density of myelinated nerve fibers (Vnerv). Twenty-six intact permanent premolars were included in the study. For histological analysis, the pulp tissue was stained with hematoxylin-eosin and immunohistochemically for von Willebrand factor and S100 to detect blood vessels and myelinated nerve fibers, respectively. The stereological analysis was used to determine the Vvasc and Vnerv. Statistical analysis was done using the Pearson correlation test and Welch’s ANOVA test. Histological analysis showed that the pulp tissue was strongly vascularized and innervated. A significant positive correlation was found between Vvasc and hemoglobin oxygen saturation levels (p = 0.030). A significant negative correlation was found between Vnerv and the lowest electrical voltage that patient felt (p = 0.033). According to the maturity of the dental apex, teeth were divided into a group with open (n = 6, OA group) and closed apex (n = 20, CA group). We found that pulps in the CA group had higher Vnerv than the OA group (p = 0.037). In contrast, there were no significant differences in Vvasc of the pulp tissue (p = 0.059), oxygen saturation (p = 0.907), or electrical voltage (p = 0.113) between both groups. We can conclude that the measurement of pulse oximetry and electrical sensibility test reflects the morphology of healthy pulp tissue independently of the maturity of the dental apex.

TTR Gly83Arg Mutation: Beyond Familial Vitreous Amyloidosis.

BackgroundGly83Arg variation is a type of TTR mutation specific to the Chinese population. Patients of hereditary transthyretin amyloidosis (ATTR) with Gly83Arg variation predominantly present with blurred vision and most of these cases are reported by ophthalmologists. There is currently no systematic assessment of extraocular features of ATTR with Gly83Arg variation.MethodsSix patients and two asymptomatic carriers with molecularly confirmed Gly83Arg variation of ATTR from three unrelated families were identified by sequencing the TTR gene. The clinical, electrophysiological, ultrasonic, and pathological data were collected and analyzed.ResultsThis study included six patients and two carriers with TTR Gly83Arg mutation, all of whom came from the Han nationality of China. The average age of onset for the six patients was 39 years, and the course of disease ranged from 5 to 19 years. All the patients started with blurred vision, which was diagnosed as vitreous opacity (VO). Most of the patients developed sensory-motor polyneuropathies over years or even more than a decade (4–15 years) after VO. However, the heterogeneity of peripheral neuropathies among these patients remained large between families. Autonomic impairment also occurred after VO, with varying degrees of abnormalities seen in the associated autonomic assessments. None of the patients had any symptoms of cardiac impairment, but abnormal results were found in examinations. A combined biopsy of the sural nerve and muscle was also performed. Nerve pathology revealed the moderately reduced myelinated nerve fiber density and muscle pathology showed predominant neurogenic impairment accompanied by possible myogenic impairment.ConclusionsThis is a detailed account of Gly83Arg mutation-related ATTR, focusing on the extraocular presentations of this special variant in Chinese. Clinical features of this variant are early-onset, ocular involvement predominance, neurological, and cardiac involvement along with the disease, and relatively long survival.

Correlation analysis of electroneuromyographic, functional-anatomical and morphometric indicators of regeneration of the injured sciatic nerve

Objective. To study the electrophysiological, functional-anatomical and morphometric indicators of the sciatic nerve regeneration after complete transection and connection of the nerve stumps with epineural sutures and adhesives. Materials and methods. The experiments were carried out on white outbred male rats. The efficiency of sciatic nerve regeneration was investigated with the standard 4-6 epineural sutures and the use of polyethylene glycol hydrogel Duraseal or Tisseel fibrin glue with 2 fixation sutures. On the 14th, 30th and 60th days after the complete transection of the sciatic nerve and subsequent connection of the nerve stumps with epineural sutures, hydrogel or fibrin glue, limb function was assessed using SFI test and ENMG. The distal segment of the nerve was sampled for electron microscopic and morphometric studies. The density of the regenerated myelinated nerve fibers was studied and a correlation analysis was performed with the results of SFI test and ENMG. Results. The use of adhesives provides a similar result of connecting the transected sciatic nerve, as in standard neurorrhaphy, and regeneration of myelinated nerve fibers in the distal nerve. The regeneration density of myelinated nerve fibers significantly increased on the 30 th and 60th days in the groups in which Duraseal hydrogel and Tisseel fibrin glue were used, without a statistically significant difference in ENMG parameters (M-response amplitude, nerve conduction velocity, latency period) and the result of SFI test. The amplitude of M-response on the 30th day was statistically significantly lower after the connection of nerve stumps with Tisseel fibrin glue in comparison with the Duraseal hydrogel, whereas on the 60th day there was no difference according to the results of electrophysiological studies. Conclusions. The efficiency of sciatic nerve regeneration after the combined connection exceeds the standard technique, and the results of pathophysiological assessments are more often correlated with morphometry data on the 30th day.

Combining chitin biological conduits with small autogenous nerves and platelet-rich plasma for the repair of sciatic nerve defects in rats.

AimsPeripheral nerve defects are often difficult to recover from, and there is no optimal repair method. Therefore, it is important to explore new methods of repairing peripheral nerve defects. This study explored the efficacy of nerve grafts constructed from chitin biological conduits combined with small autogenous nerves (SANs) and platelet‐rich plasma (PRP) for repairing 10‐mm sciatic nerve defects in rats.MethodsTo prepare 10‐mm sciatic nerve defects, SANs were first harvested and PRP was extracted. The nerve grafts consisted of chitin biological conduits combined with SAN and PRP, and were used to repair rat sciatic nerve defects. These examinations, including measurements of axon growth efficiency, a gait analysis, electrophysiological tests, counts of regenerated myelinated fibers and observations of their morphology, histological evaluation of the gastrocnemius muscle, retrograde tracing with Fluor‐Gold (FG), and motor endplates (MEPs) distribution analysis, were conducted to evaluate the repair status.ResultsTwo weeks after nerve transplantation, the rate and number of regenerated axons in the PRP‐SAN group improved compared with those in the PRP, SAN, and Hollow groups. The PRP‐SAN group exhibited better recovery in terms of the sciatic functional index value, composite action potential intensity, myelinated nerve fiber density, myelin sheath thickness, and gastrectomy tissue at 12 weeks after transplantation, compared with the PRP and SAN groups. The results of FG retrograde tracing and MEPs analyses showed that numbers of FG‐positive sensory neurons and motor neurons as well as MEPs distribution density were higher in the PRP‐SAN group than in the PRP or SAN group.ConclusionsNerve grafts comprising chitin biological conduits combined with SANs and PRP significantly improved the repair of 10‐mm sciatic nerve defects in rats and may have therapeutic potential for repairing peripheral nerve defects in future applications.

Low-Intensity Pulsed Ultrasound Prompts Both Functional and Histologic Improvements While Upregulating the Brain-Derived Neurotrophic Factor Expression after Sciatic Crush Injury in Rats

The aim of this study was to determine that low-intensity pulsed ultrasound (LIPUS) at an intensity of 140 mW/cm2 promotes functional and histologic improvements in sciatic nerve crush injury in a rat model and to investigate changes over time in relevant growth factors and receptors, exploring the mechanism of LIPUS in the recovery process after injury. Toe angle in the toe-off phase, regenerative axonal length, myelinated nerve fiber density, diameter of myelinated nerve fiber, axon diameter and myelin sheath thickness were significantly higher in the LIPUS group than in the sham group. Gene and protein expression of brain-derived neurotrophic factor (BDNF) was upregulated in the LIPUS group. In conclusion, LIPUS contributed to rapid functional and histologic improvement and upregulated BDNF expression after sciatic nerve crush injury in rats.

Scattered Light Imaging: Resolving the substructure of nerve fiber crossings in whole brain sections with micrometer resolution

For developing a detailed network model of the brain based on image reconstructions, it is necessary to spatially resolve crossing nerve fibers. The accuracy hereby depends on many factors, including the spatial resolution of the imaging technique. 3D Polarized Light Imaging (3D-PLI) allows the three-dimensional reconstruction of nerve fiber tracts in whole brain sections with micrometer in-plane resolution, but leaves uncertainties in pixels containing crossing fibers. Here we introduce Scattered Light Imaging (SLI) to resolve the substructure of nerve fiber crossings. The measurement is performed on the same unstained histological brain sections as in 3D-PLI. By illuminating the brain sections from different angles and measuring the transmitted (scattered) light under normal incidence, light intensity profiles are obtained that are characteristic for the underlying brain tissue structure. We have developed a fully automated evaluation of the intensity profiles, allowing the user to extract various characteristics, like the individual directions of in-plane crossing nerve fibers, for each image pixel at once. We validate the reconstructed nerve fiber directions against results from previous simulation studies, scatterometry measurements, and fiber directions obtained from 3D-PLI. We demonstrate in different brain samples (human optic tracts, vervet monkey brain, rat brain) that the 2D fiber directions can be reliably reconstructed for up to three crossing nerve fiber bundles in each image pixel with an in-plane resolution of up to 6.5 μm. We show that SLI also yields reliable fiber directions in brain regions with low 3D-PLI signals coming from regions with a low density of myelinated nerve fibers or out-of-plane fibers. This makes Scattered Light Imaging a promising new imaging technique, providing crucial information about the organization of crossing nerve fibers in the brain.

Conventional histomorphometry and fast free of acrylamide clearing tissue (FACT) visualization of sciatic nerve in chicken (Gallus domesticus).

Histomorphometry and use of the fast free of acrylamide clearing tissue (FACT) protocol were studied on the sciatic nerve in chicken (Gallus domesticus). In the first part of the study, the sciatic nerves of 20 chickens of four age groups (7, 14, 26 and 40 days) were studied (n=5 birds per age class). Their sciatic nerve samples were stained with Hematoxylin and Eosin and Masson's trichrome and were histomorphometrically evaluated. In the second part of the study, FACT protocol was applied on the sciatic nerve of a 26 days old chicken. After clearing of 1.00 mm-thick sciatic nerve sections, they were immunolabelled using Hoechst for nuclei staining and recorded by a Z-stack motorized fluorescent microscope. In the conventional histo-morphometry, the epineurium, perineurium and endoneurium were thicker and the nerve bundle diameter was bigger in the left sciatic nerve of chicken of all age groups compared to the right sciatic nerve. On the contrary, the axon diameter and the myelinated nerve fiber diameter were bigger, the myelin sheath was thicker, the nodes of Ranvier intervals were higher and the density of myelinated nerve fibers was also higher in the right sciatic nerve compared to the left one. In conclusion, histomorphometric parameters in the left and right sciatic nerve during chicken growth were significantly different. Furthermore, the FACT protocol could be used for the 3D imaging of the chicken sciatic nerve and its immunostained evaluation.

A novel tissue engineered nerve graft constructed with autologous vein and nerve microtissue repairs a long-segment sciatic nerve defect.

Veins are easy to obtain, have low immunogenicity, and induce a relatively weak inflammatory response. Therefore, veins have the potential to be used as conduits for nerve regeneration. However, because of the presence of venous valves and the great elasticity of the venous wall, the vein is not conducive to nerve regeneration. In this study, a novel tissue engineered nerve graft was constructed by combining normal dissected nerve microtissue with an autologous vein graft for repairing 10-mm peripheral nerve defects in rats. Compared with rats given the vein graft alone, rats given the tissue engineered nerve graft had an improved sciatic static index, and a higher amplitude and shorter latency of compound muscle action potentials. Furthermore, rats implanted with the microtissue graft had a higher density and thickness of myelinated nerve fibers and reduced gastrocnemius muscle atrophy compared with rats implanted with the vein alone. However, the tissue engineered nerve graft had a lower ability to repair the defect than autogenous nerve transplantation. In summary, although the tissue engineered nerve graft constructed with autologous vein and nerve microtissue is not as effective as autologous nerve transplantation for repairing long-segment sciatic nerve defects, it may nonetheless have therapeutic potential for the clinical repair of long sciatic nerve defects. This study was approved by the Experimental Animal Ethics Committee of Chinese PLA General Hospital (approval No. 2016-x9-07) on September 7, 2016.

879 Novel injectable coolant for reducing cutaneous nerve fiber density

Postherpetic neuralgia (PHN) is a dermatologic disorder with no established cure. The distinct dermatomal distribution of PHN makes the peripheral nerve supplying the dermatome and its distal cutaneous branches the ideal therapeutic target; however, the short acting nature of current medications limits the use of this localized approach. Herein we demonstrate the effects of a drug-free, injectable coolant with long-lasting pain suppression capabilities that could potentially be used for treatment of PHN. We have previously reported reduced cutaneous nerve fiber density following topical cooling with cryolipolysis treatment. From here we went on to develop an injectable coolant to target nerves at any anatomic location accessible by a needle. We showed that injection of coolant around the rat sciatic nerve leads to local disruption of myelin. This in turn leads to decrease in nocifensive function for up to 60 days. In this study, using the rat sciatic nerve, we examined the mechanism of reduced nociceptive response after coolant injection. We investigated the effects of the injectable coolant on sciatic nerve branches distal to the injection site and on cutaneous nerve fibers using Coherent anti-Stokes Raman scattering (CARS) microscopy and immunofluorescence (IF) staining. CARS showed myelin degradation in branches distal to the injection site. Corrected correlation parameter index, an indicator for organization of myelin structure, decreased in fibular branch from 0.86±0.08 at baseline to 0.40±0.22 (P<0.0001) at day 14 post injection. Myelinated dermal nerve fiber density was quantified in skin of the hind paw innervated by the sciatic nerve. IF staining showed decreased density of myelinated dermal nerve fibers from baseline value of 56±3/mm2 to 22±3/mm2 (P<0.0001) at day 14. Thus, coolant injection reduces pain by decreasing cutaneous nerve fiber density. Although more work needs to be done, we hope this could potentially be used to treat PHN associated pain.

Peri‐implant myelinated nerve fibers: Histological findings in dogs

While osseointegration following various dental implant placement protocols has been extensively investigated, the neurohistological integration has received little attention. The primary aim of this study was to compare the myelinated nerve fibers density in peri-implant bone tissue following various implant placement protocols. The secondary aim assessed the effect of follow-up on peri-implant nerve fibers density. Ten beagle dogs randomly received 68 commercially pure titanium implants in the mandibular premolar or molar regionbilaterally following extraction utilizingone of the six treatment protocols: (a) immediate implant placement (IIP) and immediate loading (IL); (b) IIP and delayed loading (DL); (c) IIP and left unloaded (UL); (d) delayed implant placement (DIP) and IL; (e) DIP and DL; and (f) DIP and UL. Histomorphometric analysis of the peri-implant myelinated nerve fibers was performed in a 300μm peri-implant zone at the cervical, middle, and apical level following implant placement. The follow-up assessment involved longitudinal observation at 3months following each implant treatment protocol and at 6months for IIP+IL and IIP+DL protocols. The influence of different treatment protocols, including the fixed effects of implant groups (IIP+IL, IIP+DL, IIP+UL, DIP+IL, DIP+DL, DIP+UL) and regions (cervical, middle, apical), was examined via a linear mixed model. The IIP+IL group showed a significantly higher myelinated nerve density compared to the IIP+UL and DIP+UL group. Peri-implant nerve re-innervation was significantly higher (P=.002) in the apical region compared to the cervical region. After immediate implant placement, the IL group showed a significantly (P=.03) higher density of myelinated nerve fibers compared to DL. No significant (P=.19) effect of follow-up on nerve density was observed. The immediate implant placement and loading protocol showed most beneficial effect on peri-implant innervation with highest myelinated nerve density in the apical region. A longer loading time had no influence on the peri-implant nerve density.

Platelet-Rich Plasma Combined with Low-Dose Ultrashort Wave Therapy Accelerates Peripheral Nerve Regeneration.

Finding treatments that accelerate peripheral nerve regeneration, prolongation, and functional recovery remains a challenging task. Platelet-rich plasma (PRP) contains numerous growth factors and active proteins, and low-dose ultrashort waves (USWs) stimulate the formation of nerve-nourishing vessels, which are powerful for nerve regeneration. The goal of this study was to evaluate the synergistic effects of serial ultrasound-guided PRP injections combined with low-dose USWs radiation on peripheral nerve regeneration in a crush injury model. Fifty rabbits were equally and randomly divided into normal control, model, USW, PRP, and PRP+USW groups. The neurological function, electrophysiological recovery, and histological and morphological evaluation of regenerated nerves, as well as a targeted muscle recovery assessment, were performed to investigate the regenerative effect of PRP combined with USW therapy. Our results showed that the PRP+USW group had the better early axonal regeneration and displayed the earliest positive compound muscle action potential among the treatment groups. At postintervention week 12, a histological evaluation showed similar expression of the S-100 protein in the PRP+USW and normal control groups. Moreover, the morphological assessment revealed a significant increase in the myelinated nerve fiber density and diameter and myelin sheath thickness compared with the USW and PRP groups. The morphometry of the target muscles indicated the lowest reduction in the percent volume in the PRP+USW group, and an ultrasound examination of the targeted muscle showed the best improvement in stiffness and perfusion parameters at 12 weeks after crush injury. Thus, serial ultrasound-guided PRP injections combined with low-dose USW radiation exert a synergistic effect on accelerating functional axon recovery and decreasing atrophy of the target muscles in a crush injury model. Impact Statement This research describes that the application of platelet-rich plasma combined with low-dose ultrashort waves treatment exert a synergistic effect on accelerating peripheral nerve regeneration. With the extensive use of platelet-rich plasma and physical factors in regenerative medicine or clinical rehabilitation medicine, our findings may help establish effective strategies for repairing peripheral nerve injury.

Research of acellular xenogeneic nerve combined with adipose-derived stem cells and platelet rich plasma in repair of rabbit facial nerve injury

To investigate the early effects of acellular xenogeneic nerve combined with adipose-derived stem cells (ADSCs) and platelet rich plasma (PRP) in repairing facial nerve injury in rabbits. The bilateral sciatic nerves of 15 3-month-old male Sprague-Dawley rats were harvested and decellularized as xenografts. The allogeneic ADSCs were extracted from the neck and back fat pad of healthy adult New Zealand rabbits with a method of digestion by collagenase type Ⅰ and the autologous PRP was prepared by two step centrifugation. The 3rd generation ADSCs with good growth were labelled with CM-Dil living cell stain, and the labelling and fluorescence attenuation of the cells were observed by fluorescence microscope. Another 32 New Zealand rabbits were randomly divided into 4 groups and established the left facial nerve defect in length of 1 cm ( n=8). The nerve defects of groups A, B, C, and D were repaired with CM-Dil-ADSCs composite xenogeneic nerve+autologous PRP, CM-Dil-ADSCs composite xenogeneic nerve, xenogeneic nerve, and autologous nerve, respectively. At 1 and 8 weeks after operation, the angle between the upper lip and the median line of the face (angle θ) was measured. At 4 and 8 weeks after operation, the nerve conduction velocity was recorded by electrophysiological examination. At 8 weeks after operation, the CM-Dil-ADSCs at the distal and proximal ends of regenerative nerve graft segment in groups A and B were observed by fluorescence microscopy; after toluidine blue staining, the number of myelinated nerve fibers in regenerated nerve was calculated; the structure of regenerated nerve fibers was observed by transmission electron microscope. ADSCs labelled by CM-Dil showed that the labelling rate of cells was more than 90% under fluorescence microscope, and the labelled cells proliferated well, and the fluorescence attenuated slightly after passage. All the animals survived after operation, the incision healed well and no infection occurred. At 1 week after operation, all the animals in each group had different degrees of dysfunction. The angle θ of the left side in groups A, B, C, and D were (53.4±2.5), (54.0±2.6), (53.7±2.4), and (53.0±2.1)°, respectively; showing significant differences when compared with the healthy sides ( P<0.05). At 8 weeks after operation, the angle θ of the left side in groups A, B, C, and D were (61.9±4.7), (56.8±4.2), (54.6±3.8), and (63.8±5.8)°, respectively; showing significant differences when compared with the healthy sides and with the values at 1 week ( P<0.05). Gross observation showed that the integrity and continuity of regenerated nerve in 4 groups were good, and no neuroma and obvious enlargement was found. At 4 and 8 weeks after operation, the electrophysiological examination results showed that the nerve conduction velocity was significantly faster in groups A and D than in groups B and C ( P<0.05), and in group B than in group C ( P<0.05); no significant difference was found between groups A and D ( P>0.05). At 8 weeks after operation, the fluorescence microscopy observation showed a large number of CM-Dil-ADSCs passing through the distal and proximal transplants in group A, and relatively few cells passing in group B. Toluidine blue staining showed that the density of myelinated nerve fibers in groups A and D were significantly higher than those in groups B and C ( P<0.05), and in group B than in group C ( P<0.05); no significant difference was found between groups A and D ( P>0.05). Transmission electron microscope observation showed that the myelinated nerve sheath in group D was large in diameter and thickness in wall. The morphology of myelin sheath in group A was irregular and smaller than that in group D, and there was no significant difference between groups B and C. ADSCs can survive as a seed cell in vivo, and can be differentiated into Schwann-like cells under PRP induction. It can achieve better results when combined with acellular xenogeneic nerve to repair peripheral nerve injury in rabbits.

Sensory nerve degeneration in a mouse model mimicking early manifestations of familial amyloidpolyneuropathy due to transthyretin Ala97Ser.

Sensory nerve degeneration and consequent abnormal sensations are the earliest and most prevalent manifestations of familial amyloid polyneuropathy (FAP) due to amyloidogenic transthyretin (TTR). FAP is a relentlessly progressive degenerative disease of the peripheral nervous system. However, there is a lack of mouse models to replicate the early neuropathic manifestations of FAP. We established human TTR knock-in mice by replacing one allele of the mouse Ttr locus with human wild-type TTR (hTTRwt ) or human TTR with the A97S mutation (hTTRA97S ). Given the late onset of neuropathic manifestations in A97S-FAP, we investigated nerve pathology, physiology, and behavioural tests in these mice at two age points: the adult group (8 - 56weeks) and the ageing group (>104weeks). In the adult group, nerve profiles, neurophysiology and behaviour were similar between hTTRwt and hTTRA97S mice. By contrast, ageing hTTRA97S mice showed small fibre neuropathy with decreased intraepidermal nerve fibre density and behavioural signs of mechanical allodynia. Furthermore, significant reductions in sural nerve myelinated nerve fibre density and sensory nerve action potential amplitudes in these mice indicated degeneration of large sensory fibres. The unaffected motor nerve physiology replicated the early symptoms of FAP patients, that is, sensory nerves were more vulnerable to mutant TTR than motor nerves. These results demonstrate that the hTTRA97S mouse model develops sensory nerve pathology and corresponding physiology mimicking A97S-FAP and provides a platform to develop new therapies for the early stage of A97S-FAP.

Melatonin attenuates scopolamine-induced cognitive impairment via protecting against demyelination through BDNF-TrkB signaling in the mouse dentate gyrus

Animal models of scopolamine-induced amnesia are widely used to study underlying mechanisms and treatment of cognitive impairment in neurodegenerative diseases such as Alzheimer's disease (AD). Previous studies have identified that melatonin improves cognitive dysfunction in animal models. In this study, using a mouse model of scopolamine-induced amnesia, we assessed spatial and short-term memory functions for 4 weeks, investigated the expression of myelin-basic protein (MBP) in the dentate gyrus, and examined whether melatonin and scopolamine cotreatment could keep cognitive function and MBP expression. In addition, to study functions of melatonin for keeping cognitive function and MBP expression, we examined expressions of brain-derived neurotrophic factor (BDNF) and tropomycin receptor kinase B (TrkB) in the mouse dentate gyrus. Scopolamine (1 mg/kg) and melatonin (10 mg/kg) were intraperitoneally treated for 2 and 4 weeks. Two and 4 weeks after scopolamine treatment, mice showed significant cognitive impairment; however, melatonin and scopolamine cotreatment recovered cognitive impairment. Two and 4 weeks of scopolamine treatment, the density of MBP immunoreactive myelinated nerve fibers was significantly decreased in the dentate gyrus; however, scopolamine and melatonin cotreatment significantly increased the scopolamine-induced reduction of MBP expression in the dentate gyrus. Furthermore, the cotreatment of scopolamine and melatonin significantly increased the scopolamine-induced decrease of BDNF and TrKB immunoreactivity in the dentate gyrus. Taken together, our results indicate that melatonin treatment exerts anti-amnesic effect and restores the scopolamine-induced reduction of MBP expression through increasing BDNF and TrkB expressions in the mouse dentate gyrus.