Chitosan Functionalized Magnetic Nanoparticles to Provide Neural Regeneration and Recovery after Experimental Model Induced Peripheral Nerve Injury

(1) Background: Peripheral nerve injuries have a great impact on a patient’s quality of life and a generally poor outcome regarding functional recovery. Lately, studies have focused on different types of nanoparticles and various natural substances for the treatment of peripheral nerve injuries. This is the case of chitosan, a natural compound from the crustaceans’ exoskeleton. The present study proposes to combine chitosan benefic properties to the nanoparticles’ ability to transport different substances to specific locations and evaluate the effects of magnetic nanoparticles functionalized with chitosan (CMNPs) on peripheral nerve injuries’ rehabilitation by using an in vivo experimental model. (2) Methods: CMNPs treatment was administrated daily, orally, for 21 days to rats subjected to right sciatic nerve lesion and compared to the control group (no treatment) by analyzing the sciatic functional index, pain level, body weight, serum nerve growth factor levels and histology, TEM and EDX analysis at different times during the study. (3) Results: Animals treated with CMNPs had a statistically significant functional outcome compared to the control group regarding: sciatic functional index, pain-like behavior, total body weight, which were confirmed by the histological and TEM images. (4) Conclusions: The results of the study suggest that CMNPs appear to be a promising treatment method for peripheral nerve injuries.

Effects of normal or reversed proximodistal orientation of autologous nerve grafts on functional and histomorphological outcomes

Background: Autologous nerve grafting has been considered the gold standard for the treatment of irreparable nerve gaps. However, the choice of effective proximodistal orientation of autografts (normal or reversed) is controversial. Therefore, we compared functional and histological outcomes between normal and reversed orientations of autografts.Methods: A 10-mm section of the sciatic nerve was harvested from a donor mouse. Half the harvested nerve was grafted onto an irreparable gap in a recipient mouse using either a normal or reversed orientation. The sciatic functional index (SFI) was measured serially for up to 12 weeks postoperatively. Morphological analysis was performed using immunofluorescence staining for neurofilament (NF) and myelin protein zero (P0) in cross-sectional and whole-mount nerve preparations. Additionally, morphological analysis of the tibialis anterior muscle was performed using hematoxylin and eosin staining.Results: The SFI recovered gradually up to 12 weeks after autografting, but there were no significant differences in the SFI between the normal and reversed orientations. The number of NF-expressing axons was significantly higher in the normal orientation than in the reversed orientation. However, there were no significant differences in the number and mean intensity of P0-expressing axons between the orientations. The cross-sectional area of myofibers was significantly larger in the normal orientation than in the reversed orientation.Conclusions: Normally oriented autografts promote axonal regrowth and prevent neurogenic muscular atrophy compared with reverse-oriented autografts. However, despite these positive histomorphometric effects, the proximodistal orientation of the autograft does not affect functional outcomes.

The effects of adipose-derived multipotent mesenchymal stromal cells transplantation on motor activity and function of the sciatic nerve in mice with peripheral neuropathy

The Charcot–Marie–Tooth disease type 1A (SHMT1A) is one of the most common hereditary motor-sensory peripheral neuropathies, which is caused by demyelination of motor and sensory nerve fibers and leads to nerve dysfunction. There are currently no effective treatments for hereditary neuropathies, but recent studies indicate a number of potentially effective therapeutic agents, including multipotent mesenchymal stromal cells (MMSCs). The aim of the study was to evaluate the effect of adipose-derived MMSCc transplantation on motor activity and sciatic nerve function of transgenic mice with peripheral neuropathy. Materials and methods. The transgenic B6.Cg-Tg(PMP22)C3Fbas/J mice with peripheral neuropathy were injected intramuscularly with MMSCs, which were isolated from the adipose tissue of FVB-Cg-Tg(GFPU) mice transgenic by GFP. Motor activity of experimental animals was investigated in dynamics after 2, 4, 6, 8 and 10 weeks using the behavioral balance beam test. The functions of the sciatic nerve were analyzed according to the footprint test by calculating the sciatic functional index (SFI). Results. For 2-10 weeks in animals with neuropathy, disease progression was observed, which was expressed in an increasing increase in the number of slidings of the hind limbs from the beam and the time required to walking the distance. SFI in animals of this group decreased and at the 10th week was -47.0 ± 2 units. In contrast, from the 2nd week of the experiment, mice with neuropathy after MMSCs transplantation performed 20 % fewer slidings and spent 11 % less time in the balance beam test compared to animals without cell transplantation. In the same period, an increase of SFI up to -30.2 ± 2 versus -34.6 ± 0.9 units was observed, respectively. At the 10th week after the injection of MMSC, the SFI value was -10.1 ± 2.3 units and correlated with a decrease in the number of slidings and the time spent on the balance beam test. Conclusions. MMSCs transplantation improves the sciatic functional index and fine motor skills in mice with peripheral neuropathy. MMSCs have the potential to be an effective therapeutic agent in the treatment of peripheral neuropathy at Charcot-Marie-Tooth disease.

Rapid-stretch injury to peripheral nerves: implications from an animal model

OBJECTIVERapid-stretch nerve injuries are among the most devastating lesions to peripheral nerves, yielding unsatisfactory functional outcomes. No animal model has yet been developed that uses only stretch injury for investigation of the pathophysiology of clinical traction injuries. The authors’ objective was to define the behavioral and histopathological recovery after graded rapid-stretch nerve injury.METHODSFour groups of male B6.Cg-Tg(Thy1-YFP)HJrs/J mice were tested: sham injury (n = 11); stretch within elastic limits (elastic group, n = 14); stretch beyond elastic limits but before nerve rupture (inelastic group, n = 14); and stretch-ruptured nerves placed in continuity (rupture group, n = 16). Mice were injured at 8 weeks of age, comparable with human late adolescence. Behavioral outcomes were assessed using the sciatic functional index (SFI), tapered-beam dexterity, Von Frey monofilament testing, and the Hargreaves method. Nerve regeneration outcomes were assessed by wet muscle weight and detailed nerve histology after 48 days.RESULTSPost hoc biomechanical assessment of strain and deformation confirmed that the differences between the elastic and inelastic cohorts were statistically significant. After elastic injury, there was a temporary increase in foot faults on the tapered beam (p < 0.01) and mild reduction in monofilament sensitivity, but no meaningful change in SFI, muscle weight, or nerve histology. For inelastic injuries, there was a profound and maintained decrease in SFI (p < 0.001), but recovery of impairment was observed in tapered-beam and monofilament testing by days 15 and 9, respectively. Histologically, axon counts were reduced (p = 0.04), muscle atrophy was present (p < 0.01), and there was moderate neuroma formation on trichrome and immunofluorescent imaging. Stretch-ruptured nerves healed in continuity but without evidence of regeneration. Substantial and continuous impairment was observed in SFI (p < 0.001), tapered beam (p < 0.01), and monofilament (p < 0.01 until day 48). Axon counts (p < 0.001) and muscle weight (p < 0.0001) were significantly reduced, with little evidence of axonal or myelin regeneration concurrent with neuroma formation on immunofluorescent imaging.CONCLUSIONSThe 3 biomechanical grades of rapid-stretch nerve injuries displayed consistent and distinct behavioral and histopathological outcomes. Stretch within elastic limits resembled neurapraxic injuries, whereas injuries beyond elastic limits demonstrated axonotmesis coupled with impoverished regeneration and recovery. Rupture injuries uniquely failed to regenerate, despite physical continuity of the nerve. This is the first experimental evidence to correlate stretch severity with functional and histological outcomes. Future studies should focus on the pathophysiological mechanisms that reduce regenerative capacity after stretch injury.

Closing the Gap Between Mammalian and Invertebrate Peripheral Nerve Injury: Protocol for a Novel Nerve Repair

Background Outcomes after peripheral nerve injuries are poor despite current nerve repair techniques. Currently, there is no conclusive evidence that mammalian axons are capable of spontaneous fusion after transection. Notably, certain invertebrate species are able to auto-fuse after transection. Although mammalian axonal auto-fusion has not been observed experimentally, no mammalian study to date has demonstrated regenerating axolemmal membranes contacting intact distal segment axolemmal membranes to determine whether mammalian peripheral nerve axons have the intrinsic mechanisms necessary to auto-fuse after transection. Objective This study aims to assess fusion competence between regenerating axons and intact distal segment axons by enhancing axon regeneration, delaying Wallerian degeneration, limiting the immune response, and preventing myelin obstruction. Methods This study will use a rat sciatic nerve model to evaluate the effects of a novel peripheral nerve repair protocol on behavioral, electrophysiologic, and morphologic parameters. This protocol consists of a variety of preoperative, intraoperative, and postoperative interventions. Fusion will be assessed with electrophysiological conduction of action potentials across the repaired transection site. Axon-axon contact will be assessed with transmission electron microscopy. Behavioral recovery will be analyzed with the sciatic functional index. A total of 36 rats will be used for this study. The experimental group will use 24 rats and the negative control group will use 12 rats. For both the experimental and negative control groups, there will be both a behavior group and another group that will undergo electrophysiological and morphological analysis. The primary end point will be the presence or absence of action potentials across the lesion site. Secondary end points will include behavioral recovery with the sciatic functional index and morphological analysis of axon-axon contact between regenerating axons and intact distal segment axons. Results The author is in the process of grant funding and institutional review board approval as of March 2020. The final follow-up will be completed by December 2021. Conclusions In this study, the efficacy of the proposed novel peripheral nerve repair protocol will be evaluated using behavioral and electrophysiologic parameters. The author believes this study will provide information regarding whether spontaneous axon fusion is possible in mammals under the proper conditions. This information could potentially be translated to clinical trials if successful to improve outcomes after peripheral nerve injury. International Registered Report Identifier (IRRID) PRR1-10.2196/18706

Comparative Behavioral Assessment of Lewis and Nude Rats after Peripheral Nerve Injury

Cell therapy has shown potential in the field of peripheral nerve repair, and research using rodents is a critical and essential step toward clinical development of this approach. Traditionally, most experimental peripheral nerve injuries are conducted in inbred Lewis or outbred Sprague–Dawley strains. However, transplantation of xenogeneic cells such as human-derived cells typically triggers rejection in these animals. An alternative approach is to use immunodeficient animals, such as athymic nude rats. The lack of functional T cells in these animals renders them more accommodating to foreign cells from a different host. Currently, no literature exists regarding sensorimotor behavioral assessment of nude rats after peripheral nerve injury. To this end, we compared the functional recovery during a 6-wk period of behavioral testing of Lewis and nude rats after unilateral sciatic nerve crushing injury. Three sensorimotor behavioral assessments were performed weekly: a ladder rungwalking task to assess slip ratio and cross duration, von Frey nociception testing to determine the paw withdrawal threshold thus monitoring the regaining of sensory function, and sciatic functional index evaluation to monitor the recovery of integrated motor function. Both strains demonstrated significant sensory and motor deficits in the first week after injury, with a slight regain of sensory function, reduced slip ratio, and increased sciatic functional index starting at 2 wk. No significance difference existed between nude and Lewis rats in their recovery courses. We conclude that nude rats are a suitable model for behavioral training and assessment for cell transplantation studies in peripheral nerve injury and repair.

Closing the Gap Between Mammalian and Invertebrate Peripheral Nerve Injury: Protocol for a Novel Nerve Repair (Preprint)

BACKGROUND Outcomes after peripheral nerve injuries are poor despite current nerve repair techniques. Currently, there is no conclusive evidence that mammalian axons are capable of spontaneous fusion after transection. Notably, certain invertebrate species are able to auto-fuse after transection. Although mammalian axonal auto-fusion has not been observed experimentally, no mammalian study to date has demonstrated regenerating axolemmal membranes contacting intact distal segment axolemmal membranes to determine whether mammalian peripheral nerve axons have the intrinsic mechanisms necessary to auto-fuse after transection. OBJECTIVE This study aims to assess fusion competence between regenerating axons and intact distal segment axons by enhancing axon regeneration, delaying Wallerian degeneration, limiting the immune response, and preventing myelin obstruction. METHODS This study will use a rat sciatic nerve model to evaluate the effects of a novel peripheral nerve repair protocol on behavioral, electrophysiologic, and morphologic parameters. This protocol consists of a variety of preoperative, intraoperative, and postoperative interventions. Fusion will be assessed with electrophysiological conduction of action potentials across the repaired transection site. Axon-axon contact will be assessed with transmission electron microscopy. Behavioral recovery will be analyzed with the sciatic functional index. A total of 36 rats will be used for this study. The experimental group will use 24 rats and the negative control group will use 12 rats. For both the experimental and negative control groups, there will be both a behavior group and another group that will undergo electrophysiological and morphological analysis. The primary end point will be the presence or absence of action potentials across the lesion site. Secondary end points will include behavioral recovery with the sciatic functional index and morphological analysis of axon-axon contact between regenerating axons and intact distal segment axons. RESULTS The author is in the process of grant funding and institutional review board approval as of March 2020. The final follow-up will be completed by December 2021. CONCLUSIONS In this study, the efficacy of the proposed novel peripheral nerve repair protocol will be evaluated using behavioral and electrophysiologic parameters. The author believes this study will provide information regarding whether spontaneous axon fusion is possible in mammals under the proper conditions. This information could potentially be translated to clinical trials if successful to improve outcomes after peripheral nerve injury. INTERNATIONAL REGISTERED REPORT PRR1-10.2196/18706

PENGGUNAAN SCIATIC FUNCTIONAL INDEX (SFI) DAN TIBIAL FUNCTIONAL INDEX (TFI) DALAM PENILAIAN REGENERASI MOTORIS SARAF TEPI

Regenerasi motorik saraf tepi dapat dinilai dengan menggunakan Sciatic Function Index (SFI) dan Tibial Function Index (TFI). Namun, studi komprehensif mengenai aspek anatomi dan metodologis pengukuran belum memadai. Penelitian ini bertujuan membandingkan penggunaan TFI dan SFI dalam menilai pemulihan fungsi motorik saraf tepi. Penelitian ini menggunakan desain posttest only with control group. Delapan belas tikus Wistar jantan dibagi menjadi tiga kelompok. Kelompok I, nervus ischiadicus dicederai 5mm proksimal terhadapat bifurkasi nervus ischiadicus kaki belakang kanan. Kelompok II, nervus tibialis dicederai 5mm proksimal dari tempat bercabang nervus cutaneous surae medialis kaki belakang kanan dan kelompok III sebagai kontrol negatif. SFI dan TFI diamati pada minggu II, IV, VI, dan VIII dan dianalisis. Hasil penelitian ini menunjukkan terdapat perbedaan yang signifikan antara nilai varians SFI dan TFI pada pengamatan minggu III, IV, dan VI (p-value < 0,05). Penggunaan TFI sebagai metode penilaian regenerasi motorik saraf tepi lebih baik dari pada penggunaan SFI yang ditunjukkan oleh nilai varians yang lebih kecil.

Functional Evaluation of Sciatic Nerve Anastomosis Wraped by Freeze Dried Human Amniotic Membrane in Sprague Dawley Rat

Background. Peripheral nerve injury (PNI) is a common medical condition. The defected nerve, if not repaired as early as possible, can cause long-term denervation and neurotrophy failure for the target organ. This leads to a series of denervation manifestations, such as muscle atrophy, loss of sensory function, etc. and ultimately, these manifestations seriously affect the patient’s sensorimotor function.1,2 Amniotic membranes have been widely used in ophthalmology and skin injury repair because of their anti-inflammatory properties. In this study, we measured therapeutic efficacy and determined if amniotic membranes could be used for sciatic nerve repair.Material and methods. A post test only control group design has been done in 10 healthy Sprague Dawley rats. In all rats, a unilateral right side sciatic nerve transection was performed and reanastomosed by different methods: Group I (control group): included 5 rats, the anastomosis was done by epineural microsutures using 8/0 nylon. Group II: included 5 rats, the anastomosis was done by epineural microsutures using 8/0 nylon and then wraped by freeze dried human amniotic membrane. Functional evaluation of nerve recovery was done over 3 weeks postoperatively using walking tract analysis and calculate using Sciatic Functional Index.Result. Functional results showed that there was no significant difference of the sciatic functional index (SFI) between group I and group II.Conclusion. We can conclude that during 3 weeks functional evaluation, there is no significant difference between control group and experimental group that achieved freeze dried human amniotic membrane.Keywords: sciatic nerve injury, freeze dried human amniotic membrane, walking tract analysis, sciatic functional index.

THE ACTION OF SERICIN PROTEIN ON INITIAL NERVE REPAIR, ASSOCIATED OR NOT WITH SWIMMING IN WISTAR RATS

ABSTRACT Objective: To analyze the effects of sericin treatment, associated or not with swimming with load exercise, on initial sciatic nerve repair after compression in Wistar rats. Methods: Forty animals were divided into five groups: control, injury, injury-sericin, injury-swimming and injury-sericin-swimming. During the axonotmesis procedure, the sericin was applied to the injury-sericin and injury-sericin-swimming groups. The injury-swimming and injury-sericin-swimming groups performed the swimming with load exercise for five days, beginning on the third postoperative day (PO), and were evaluated for function, nociception and allodynia. Euthanasia was performed on the 8th PO day and fragments of the nerve were collected and prepared for quantitative and descriptive analysis in relation to the total amount of viable nerve fibers and non-viable nerve fibers, nerve fiber diameter, axon diameter and myelin sheath thickness. Results: There was no significant improvement in the sciatic functional index up to the eighth day. The Von Frey test of the surgical scar and plantar fascia indicated a reduction in pain and allodynia for the injury-swimming and injury-sericin-swimming groups. The morphological analysis presented similar characteristics in the injury-sericin, injury-swimming and injury-sericin-swimming groups, but there was a significant difference in the number of smaller non-viable nerve fibers in the injury-swimming and injury-sericin-swimming groups as compared to the others. Conclusions: Isolated sericin protein presented proinflammatory characteristics. There was improvement of allodynia and a decrease in the pain at the site of the surgical incision, possibly linked to an aquatic effect. There was no acceleration of nerve repair on the eighth day after the injury. Level of Evidence I; High quality randomized clinical trial with or without statistically significant difference, but with narrow confidence intervals.