Facial Nerve Repair by Muscle-Vein Conduit in Rats: Functional Recovery and Muscle Reinnervation.

The facial nerve is the most frequently damaged nerve in head and neck traumata. Repair of interrupted nerves is generally reinforced by fine microsurgical techniques; nevertheless, regaining all functions is the exception rather than the rule. The so-called "postparalytic syndrome," which includes synkinesia and altered blink reflexes, follows nerve injury. The purpose of this study was to examine if nerve-gap repair using an autologous vein filled with skeletal muscle would improve axonal regeneration, reduce neuromuscular junction polyinnervation, and improve the recovery of whisking in rats with transected and sutured right buccal branches of the facial nerve. Vibrissal motor performance was studied with the use of a video motion analysis. Immunofluorescence was used to visualize and analyze target muscle reinnervation. The results taken together indicate a positive effect of muscle-vein-combined conduit (MVCC) on the improvement of the whisking function after reparation of the facial nerve in rats. The findings support the recent suggestion that a venal graft with implantation of a trophic source, such as autologous denervated skeletal muscle, may promote the monoinnervation degree and ameliorate coordinated function of the corresponding muscles.

FGF-2 is required to prevent astrogliosis in the facial nucleus after facial nerve injury and mechanical stimulation of denervated vibrissal muscles.

Recently, we have shown that manual stimulation of paralyzed vibrissal muscles after facial-facial anastomosis reduced the poly-innervation of neuromuscular junctions and restored vibrissal whisking. Using gene knock outs, we found a differential dependence of manual stimulation effects on growth factors. Thus, insulin-like growth factor-1 and brain-derived neurotrophic factor are required to underpin manual stimulation-mediated improvements, whereas FGF-2 is not. The lack of dependence on FGF-2 in mediating these peripheral effects prompted us to look centrally, i.e. within the facial nucleus where increased astrogliosis after facial-facial anastomosis follows "synaptic stripping". We measured the intensity of Cy3-fluorescence after immunostaining for glial fibrillary acidic protein (GFAP) as an indirect indicator of synaptic coverage of axotomized neurons in the facial nucleus of mice lacking FGF-2 (FGF-2-/- mice). There was no difference in GFAP-Cy3-fluorescence (pixel number, gray value range 17-103) between intact wildtype mice (2.12±0.37×107) and their intact FGF-2-/- counterparts (2.12±0.27×107) nor after facial-facial anastomosis +handling (wildtype: 4.06±0.32×107; FGF-2-/-: 4.39±0.17×107). However, after facial-facial anastomosis, GFAP-Cy3-fluorescence remained elevated in FGF-2-/--animals (4.54±0.12×107), whereas manual stimulation reduced the intensity of GFAP-immunofluorescence in wild type mice to values that were not significantly different from intact mice (2.63±0.39×10). We conclude that FGF-2 is not required to underpin the beneficial effects of manual stimulation at the neuro-muscular junction, but it is required to minimize astrogliosis in the brainstem and, by implication, restore synaptic coverage of recovering facial motoneurons.

Does pulsed magnetic field therapy influence nerve regeneration in the median nerve model of the rat?

The aim of this study was to evaluate the impact of pulsed magnetic field therapy on peripheral nerve regeneration after median nerve injury and primary coaptation in the rat. Both median nerves were surgically exposed and denervated in 24 female Wistar rats. A microsurgical coaptation was performed on the right side, whereas on the left side a spontaneous healing was prevented. The study group underwent a daily pulsed magnetic field therapy; the other group served as a control group. The grasping force was recorded 2 weeks after the surgical intervention for a period of 12 weeks. The right median nerve was excised and histologically examined. The histomorphometric data and the functional assessments were analyzed by t-test statistics and one-way ANOVA. One-way ANOVA indicated a statistically significant influence of group affiliation and grasping force (P = 0.0078). Grasping strength was higher on a significant level in the experimental group compared to the control group permanently from the 9th week to the end of the study. T-test statistics revealed a significantly higher weight of the flexor digitorum sublimis muscle (P = 0.0385) in the experimental group. The histological evaluation did not reveal any statistically significant differences concerning the histomorphometric parameters. Our results suggest that the pulsed magnetic field therapy has a positive influence on the functional aspects of neural regeneration. More studies are needed to precisely evaluate and optimize the intensity and duration of the application.

Sensoric protection after median nerve injury: babysitter-procedure prevents muscular atrophy and improves neuronal recovery.

The babysitter-procedure might offer an alternative when nerve reconstruction is delayed in order to overcome muscular atrophy due to denervation. In this study we aimed to show that a sensomotoric babysitter-procedure after median nerve injury is capable of preserving irreversible muscular atrophy. The median nerve of 20 female Wistar rats was denervated. 10 animals received a sensory protection with the N. cutaneous brachii. After six weeks the median nerve was reconstructed by autologous nerve grafting from the contralateral median nerve in the babysitter and the control groups. Grasping tests measured functional recovery over 15 weeks. At the end of the observation period the weight of the flexor digitorum sublimis muscle was determined. The median nerve was excised for histological examinations. Muscle weight (P < 0.0001) was significantly superior in the babysitter group compared to the control group at the end of the study. The histological evaluation revealed a significantly higher diameter of axons (P = 0.0194), nerve fiber (P = 0.0409), and nerve surface (P = 0.0184) in the babysitter group. We conclude that sensory protection of a motor nerve is capable of preserving muscule weight and we may presume that metabolism of the sensory nerve was sufficient to keep the target muscle's weight and vitality.

Correlation analysis of histomorphometry and motor neurography in the median nerve rat model.

OBJECTIVE: Standard methods to evaluate the functional regeneration after injury of the rat median nerve are insufficient to identify any further differences of axonal nerve regeneration after restitution of motor recovery is completed. An important complementary method for assessing such differences is a histomorphometric analysis of the distal to lesion nerve fibers. Recently, an electrophysiological method has been proposed as a sensitive method to examine the quality of axonal nerve regeneration. METHODS: A linear regression analysis has been performed to correlate histomorphometric and neurographic data originating from 31 rats subjected to neurotmesis and immediate reconstruction of their right median nerve. RESULTS: A significant linear correlation between the velocity of neuromuscular conduction and the total number of nerve fibers (P = .037) as well as between the amplitude of compound muscle action potential and the total number of nerve fibers (P = .026) has been identified. Interestingly, a significant correlation between the velocity of neuromuscular conduction and the square root of the cross-sectional area of the nerve could be found (P = .008). This corresponds to a linear correlation between the velocity of neuromuscular conduction and the radius of the nerve. CONCLUSION: These results contribute in a better interpretation of morphological predictors of nerve regeneration and verify the previously described electrophysiological assessment in the median nerve rat model as a valid method.

Sensory reinnervation of free flaps in reconstruction of the breast and the upper and lower extremities.

There is long-standing debate about sensate versus non-sensate free microvascular flaps among microsurgeons. The principle of connecting not only the vascular supply, but also sensitive nerves, in free tissue transfer is attractive. However, increased operating time and partial spontaneous innervation led to the common decision to restrict microsurgical tissue transfer to the vascular anastomosis and to leave the nerves "untreated". Nevertheless, in special cases such as breast reconstruction or extremity reconstruction, the question about sensory nerve coaptation of the flaps remains open. We present our experience with free microvascular tissue transfer for breast and extremity reconstruction and compare the data with previous literature and conclude that most free flap surgeries do not benefit from nerve coaptation.

Bioartificial reconstruction of peripheral nerves using the rat median nerve model.

Different bioartificial tubes were recommended for peripheral nerve reconstruction in the past. In order to replace autologous nerve grafts this materials are still under review in different animal studies. Most of them are dealing with the rodent peripheral nerves. One very popular animal model to study different materials is the rat median nerve model. With its easy excess, simple behavioral tests and reliable long term results it is attractive to many scientists in this field. This review gives an overview about the past, current and future options in this model for bioartificial nerve tubes. It summarizes the evolution of successful implantation of different materials across short nerve gaps and demonstrates the obstacles arising from long nerve gaps and the problems associated to them.

Application of two different hemostatic procedures during microsurgical median nerve reconstruction in the rat does not hinder axonal regeneration.

BACKGROUND: Hemostatic procedures by means of electrical coagulation or application of topical agents are widely used to avoid postoperative bleeding during microsurgical reconstruction of peripheral nerves. It is speculated, however, that extensive hemostasis could provoke significant nerve damage. OBJECTIVE: To examine the effects of 2 intraoperative hemostatic procedures on peripheral nerve regeneration. METHODS: In 36 adult rats divided into 3 groups, the median nerve was transected and repaired by end-to-end neurorrhaphy. During surgery, bleeding was treated in groups 2 and 3 by application of either the topical hemostatic agent Floseal or routine bipolar coagulation. The degree of nerve regeneration was assessed in terms of motor function recovery using weekly grasping test evaluation for 3 months, muscle mass recovery of flexor digitorum sublimis, and stereological assessment of myelinated axon regeneration. RESULTS: Neither of the 2 applied hemostatic methods induced any negative effects on nerve regeneration as defined by grip strength, muscle mass recovery, and morphology of myelinated nerve fibers. In contrast, Floseal-treated animals showed a faster progression of motor function recovery, and animals subjected to bipolar coagulation revealed a higher muscle mass recovery compared with the control group. CONCLUSION: Controlled application of bipolar coagulation or Floseal does not exert a negative effect on the nerve regeneration outcome in rats. We propose that these methods can be applied safely to patients.

TRPV channel expression in human skin and possible role in thermally induced cell death.

Cell death via necrosis and apoptosis is a hallmark of deep dermal to full-thickness cutaneous burn injuries. Keratinocytes might act as thermosensory cells that transmit information regarding ambient temperature via heat-gated transient receptor potential vanilloid (TRPV) ion channels. The aim of this study was to investigate the distribution of TRPV1, 2, 3, and 4 in uninjured and thermally burned skin. The authors investigated warmth-evoked currents in keratinocytes and cell kinetics of thermally injured keratinocytes in culture with agonists and antagonists of TRPV channels. Specimens of uninjured normal skin and discarded tissue of thermally injured skin were stained for TRPV1, 2, 3, and 4. Cultured primary human keratinocytes were heated for 5 minutes at the following temperatures: 37°C (control), 42°C, and 60°C and thereafter cultured for 24 or 48 hours at 37°C. Thermally stressed cells were treated with TRPV antagonists capsazepine or ruthenium red, and cell viability capacity was determined. TRPV1, TRPV2, TRPV3, and TRPV4 immunoreactivity was differentially identified on basal and suprabasal keratinocytes of healthy human skin. Patch clamp analysis showed a functional response of human keratinocytes at temperatures >40°C. Cell death of keratinocytes after heating at 42°C was reduced by 15 and 5% with ruthenium red and by 20 and 30% by capsazepine at 24 and 48 hours, respectively. Cell death after treatment at 60°C was significantly reduced at 24 hours with capsazepine (22%) or ruthenium red (18%) but only minimally affected after 48 hours postinjury. Interaction with TRPV channels on keratinocytes may offer a new strategy to counteract cell death after thermal injury.

Immediate reconstruction of complex hand trauma with iliac crest bone graft and 2 pedicled fasciocutaneous skin flaps: a case report.

OBJECTIVE: The decision about primary or staged reconstruction of all structures in severe hand injuries is controversial. The purpose of this case report is to present a surgical protocol that lead to good functional results and rapid recovery after primary bone grafting with pedicled flap coverage of a complex hand injury. METHODS: A case is reported in which 2 iliac crest bone grafts, an extended dorsal metacarpal artery flap and a heterodigital island flap, were performed at primary intervention to reconstruct the index and middle fingers of a 17-year-old patient. RESULTS: Length and sensation of the digits were fully preserved while the destroyed joints were fused and covered with mobile soft tissue. Hereby, a satisfactory pinch grip and hand closure was restored, allowing the patient to return to work after only 2 months. CONCLUSIONS: Given proper planning and adequate debridement, primary reconstruction of all injured structures should be considered when dealing with complex hand injuries.

One-stage ear reconstruction after avulsion injury, using the amputated cartilage and a retroauricular transposition flap.

INTRODUCTION: Auricular reattachment or reconstruction after traumatic ear loss remains a challenge for the plastic reconstructive surgeon. Because of the diverse accident mechanisms, no standard algorithms exist and several modalities have been proposed in the literature. METHODS: A case of an innovative ear reconstruction of a partially avulsed ear is presented. The amputated cartilage was reattached after being deepithelized from the anterior skin. A transauricular-retroauricular random pattern flap was then harvested and used for anterior skin coverage. RESULTS: The described technique provided a nice final result without the need of any further operations. CONCLUSION: In general, a microsurgical replantation should be applied when the circumstances allow. In other cases, especially in partial upper-ear amputations with severe skin contusions, the described technique should be considered as a safe, single-step approach with good final results.

Non-viral genetic transfection of rat Schwann cells with FuGENE HD© lipofection and AMAXA© nucleofection is feasible but impairs cell viability.

PURPOSE: To determine transfection efficiency of FuGENE HD© lipofection and AMAXA© nucleofection on rat Schwann cells (SC). METHODS: The ischiadic and median nerves of 6-8 week old Lewis rats were cultured in modified melanocyte-growth medium. SCs were genetically transfected with green fluorescent protein (GFP) as reporter gene using FuGENE HD© lipofection and AMAXA© nucleofection. Transfection rates were determined by visualization of GFP fluorescence under fluorescence microscopy and cell counting. Transfected cell to non-transfected cell relation was determined. RESULTS: Purity of Schwann cell culture was 88% as determined by immunohistologic staining. Transfection rate of FuGENE HD© lipofection was 2%, transfection rate of AMAXA© nucleofection was 10%. With both methods, Schwann cells showed pronounced aggregation behavior which made them unfeasible for further cultivation. Settling of Schwann cells on laminin and poly-L-ornithine coated plates was compromised by either method. CONCLUSION: Non-viral transfection of rat SC with FuGENE HD© lipofection and AMAXA© nucleofection is basically possible with a higher transfection rate for nucleofection than for lipofection. As cell viability is compromised by either method however, viral transfection is to be considered if higher efficiency is required.

Efficacy of various durations of in vitro predegeneration on the cell count and purity of rat Schwann-cell cultures.

The efficacy of Schwann-cell cultivation can be enhanced by in vitro predegeneration of the harvested cells compared to immediate culture. The aim of this study was to improve Schwann-cell culture efficacy by comparing three different durations of predegeneration. The sciatic and median nerves of 6-8-week-old Lewis rats were harvested and subjected to either 2-day, 7-day, or 14-day predegeneration in Dulbecco's Modified Eagle's Medium supplemented with 10% fetal calf serum and 1% Penicillin/Streptomycin. Afterward, tissue was enzymatically dissociated and placed in a modified melanocyte growth medium. The cell count was determined immediately after dissociation while the cell purity was determined one subculture/trypsinization cycle later after cell attachment to the culture plate by means of optical microscopy and immunocytochemistry. Particular attention was then paid to the Schwann-cell-to-fibroblast relation. The cumulative cell count in the culture was 5.8 x 10(5) for 2-day, 1.12 x 10(6) for 7-day, and 1.48 x 10(6) for 14-day predegeneration. The culture purity was approximately equal for 2- and 7-day predegeneration (88% Schwann cells, 12% fibroblasts after 2 days; 85% Schwann cells, 15% fibroblasts after 7 days). After 14 days, however, cell cultures were significantly debased by fibroblast proliferation (57% Schwann cells, 43% fibroblasts). In vitro predegeneration is a particularly suitable procedural method to increase the cultural Schwann-cell yield. The number of cultivated rat Schwann cells is doubled by 7-day in vitro predegeneration in comparison to 2-day predegeneration. After 14-day predegeneration, however, the culture is significantly debased by fibroblasts. Therefore, 7-day in vitro predegeneration is an advisable predegeneration period.

Functional recovery after implantation of artificial nerve grafts in the rat- a systematic review.

PURPOSE: The aim of this study was to compare functional data of different nerve-gap bridging materials evaluated in rat experiments by means of a systematic review. MATERIALS AND METHODS: A systematic review was conducted, searching MEDLINE, HTS and CENTRAL to identify all trials evaluating functional recovery of artificial nerve conduits in the rat model. RESULTS: There was a trend towards a favourable outcome of conduits coated with Schwann-cells compared to the plain synthetics. Histomorphometry, electrophysiology and muscle-weight correlated poorly with functional outcome. CONCLUSION: Schwann-cell coated conduits showed promising results concerning functional recovery. Further standardization in outcome reporting is encouraged.

Chapter 19: The role of collagen in peripheral nerve repair.

Collagens are extracellular proteins characterized by a triple helical structure and predominantly involved in the formation of fibrillar and microfibrillar networks of the extracellular matrix and basement membranes. There are 29 collagen types which differ in size, structure, and function. In the peripheral nervous system, two classes of collagen molecules are expressed: fibril forming collagens (type-I, III, and V) and basement membrane collagens (type-IV). Collagens are required for normal extracellular matrix assembly and play an important role in the regulation of Schwann cell function. After injury collagen production in the severed nerve often exceeds the ideal response which is suggested to hinder the growth of sprouting axons into the appropriate distal fascicles and therefore delays and limits nerve regeneration. Both surgical techniques and pharmacological agents are developed to reduce injury induced scarring but despite this nerve regeneration is frequently incomplete. The aim of the present review is to provide the reader a clear overview of the current knowledge with respect to collagens in the peripheral nervous system and to emphasize its role after nerve injury.

Chapter 23: Manual stimulation of target muscles has different impact on functional recovery after injury of pure motor or mixed nerves.

Direct coaptation and interpositional nerve grafting (IPNG) of an injured peripheral nerve is still associated with poor functional recovery. Main reasons for that are thought to be an extensive collateral axonal branching at the site of transection and the polyinnervation of motor endplates due to terminal axonal and intramuscular sprouting. Moreover, severe changes occurring within the muscle after long-term denervation, like loss of muscle bulk and circulation as well as progressive fibrosis, have a negative effect on the quality of functional recovery after reinnervation. We have recently shown that manual stimulation (MS) of paralyzed vibrissal muscles in rat promotes full recovery after facial nerve coaptation. Furthermore, MS improved functional recovery after hypoglossal nerve repair, hypoglossal-facial IPNG of the facial nerve in rat. In contrary, MS did not improve recovery after injury of the median nerve in rat, which is however a mixed peripheral nerve comparing to the facial nerve. It is speculated that manually stimulated recovery of motor function requires an intact sensory input, which is affected in case of mixed peripheral nerves but not in case of pure motor nerves. In this article, we summarize our results of MS in several peripheral nerve injury models in order to illustrate the application potential of this method and to give insights into further investigations on that field.

Electrical stimulation of paralyzed vibrissal muscles reduces endplate reinnervation and does not promote motor recovery after facial nerve repair in rats.

The outcome of peripheral nerve injuries requiring surgical repair is poor. Recent work has suggested that electrical stimulation (ES) of denervated muscles could be beneficial. Here we tested whether ES has a positive influence on functional recovery after injury and surgical repair of the facial nerve. Outcomes at 2 months were compared to animals receiving sham stimulation (SS). Starting on the first day after end-to-end suture (facial-facial anastomosis), electrical stimulation (square 0.1 ms pulses at 5 Hz at an ex tempore established threshold amplitude of between 3.0 and 5.0V) was delivered to the vibrissal muscles for 5 min a day, 3 times a week. Restoration of vibrissal motor performance following ES or SS was evaluated using the video-based motion analysis and correlated with the degree of collateral axonal branching at the lesion site, the number of motor endplates in the target musculature and the quality of their reinnervation, i.e. the degree of mono- versus poly-innervation. Neither protocol reduced collateral branching. ES did not improve functional outcome, but rather reduced the number of innervated motor endplates to approximately one-fifth of normal values and failed to reduce the proportion of poly-innervated motor endplates. We conclude that ES is not beneficial for recovery of whisker function after facial nerve repair in rats.