The Use of a Hypoallergenic Dermal Matrix for Wrapping in Peripheral Nerve Lesions Regeneration: Functional and Quantitative Morphological Analysis in an Experimental Animal Model.

INTRODUCTION: The aim of this research was to test, in an animal model, the nerve regeneration technique with a hypoallergenic acellular dermal matrix used to wrap the microsurgical neural suture. MATERIALS AND METHODS: Two groups of rats received the cut of limb right median nerves. The regeneration technique considers for both groups an end-to-end nerve suture. In the experimental group (A) was used also a wrapping protocol by a conduit of collagen matrix currently used in oral surgery. The animals underwent functional grasping tests (at 1, 3, 5, and 7 months) and a histological and quantitative analysis of distal nerve was performed at the end of experimental time. RESULT: After seven months, the grasping test reveals functional recovery in each tested animal; this improvement is more evident in Group A. The fibers appear well organized with restored myelin sheaths in both groups. Group A showed a great quantity of connective tissue surrounding the nerve. The quantitative morphology analysis in both groups shows a similar fibers density, fiber diameter, and myelin thickness. The differences between the groups in axon mean diameter are significant. In Group A M/d, D/d, and g-ratio is significantly higher compared to control group. CONCLUSIONS: Histological and functional assessments show a functional recovery of the injured nerve in the test groups, stressed by the results of the grasping tests and the meaningful increasing in fiber diameter and higher g-ratio. Moreover, a connective tissue cuff distinguishes the distal portion of the injured nerve. Considering the easy availability and handling of the material used in this study we can conclude that this experimental technique can be considered as a valid alternative to protect nerves in nerve wrap surgery.

The reasons for end-to-side coaptation: how does lateral axon sprouting work?

Nerve fibers are attracted by sutureless end-to-side nerve coaptation into the recipient nerve. Opening a window in the epineurium enhances axon attraction and myelination. The authors analyze the features of nerve repair by end-to-side coaptation. They highlight the known mechanisms of axon sprouting and different hypotheses of start up signals (presence or absence of an epineurial window, role of Schwann cells, signaling from the distal trunk). The clinical literature is also presented and differences between experimental and clinical applications are pointed out. The authors propose their point of view and perspectives deriving from recent experimental and clinical experiences.

Epineurial Window Is More Efficient in Attracting Axons than Simple Coaptation in a Sutureless (Cyanoacrylate-Bound) Model of End-to-Side Nerve Repair in the Rat Upper Limb: Functional and Morphometric Evidences and Review of the Literature.

End-to-side nerve coaptation brings regenerating axons from the donor to the recipient nerve. Several techniques have been used to perform coaptation: microsurgical sutures with and without opening a window into the epi(peri)neurial connective tissue; among these, window techniques have been proven more effective in inducing axonal regeneration. The authors developed a sutureless model of end-to-side coaptation in the rat upper limb. In 19 adult Wistar rats, the median and the ulnar nerves of the left arm were approached from the axillary region, the median nerve transected and the proximal stump sutured to the pectoral muscle to prevent regeneration. Animals were then randomly divided in two experimental groups (7 animals each, 5 animals acting as control): Group 1: the distal stump of the transected median nerve was fixed to the ulnar nerve by applying cyanoacrylate solution; Group 2: a small epineurial window was opened into the epineurium of the ulnar nerve, caring to avoid damage to the nerve fibres; the distal stump of the transected median nerve was then fixed to the ulnar nerve by applying cyanoacrylate solution. The grasping test for functional evaluation was repeated every 10-11 weeks starting from week-15, up to the sacrifice (week 36). At week 36, the animals were sacrificed and the regenerated nerves harvested and processed for morphological investigations (high-resolution light microscopy as well as stereological and morphometrical analysis). This study shows that a) cyanoacrylate in end-to-side coaptation produces scarless axon regeneration without toxic effects; b) axonal regeneration and myelination occur even without opening an epineurial window, but c) the window is related to a larger number of regenerating fibres, especially myelinated and mature, and better functional outcomes.

Repairing nerve gaps by vein conduits filled with lipoaspirate-derived entire adipose tissue hinders nerve regeneration.

In spite of great recent advancements, the definition of the optimal strategy for bridging a nerve defect, especially across long gaps, still remains an open issue since the amount of autologous nerve graft material is limited while the outcome after alternative tubulization techniques is often unsatisfactory. The aim of this study was to investigate a new tubulization technique based on the employment of vein conduits filled with whole subcutaneous adipose tissue obtained by lipoaspiration. In adult rats, a 1cm-long defect of the left median nerve was repaired by adipose tissue-vein-combined conduits and compared with fresh skeletal muscle tissue-vein-combined conduits and autologous nerve grafts made by the excised nerve segment rotated by 180°. Throughout the postoperative period, functional recovery was assessed using the grasping test. Regenerated nerve samples were withdrawn at postoperative month-6 and processed for light and electron microscopy and stereology of regenerated nerve fibers. Results showed that functional recovery was significantly slower in the adipose tissue-enriched group in comparison to both control groups. Light and electron microscopy showed that a large amount of adipose tissue was still present inside the vein conduits at postoperative month-6. Stereology showed that all quantitative morphological predictors analyzed performed significantly worse in the adipose tissue-enriched group in comparison to the two control groups. On the basis of this experimental study in the rat, the use of whole adipose tissue for tissue engineering of peripheral nerves should be discouraged. Pre-treatment of adipose tissue aimed at isolating stromal vascular fraction and/or adipose derived stem/precursor cells should be considered a fundamental requisite for nerve repair.

Direct muscle neurotization after end-to end and end-to-side neurorrhaphy: An experimental study in the rat forelimb model.

The need for the continuous research of new tools for improving motor function recovery after nerve injury is justified by the still often unsatisfactory clinical outcome in these patients. It has been previously shown that the combined use of two reconstructive techniques, namely end-to-side neurorrhaphy and direct muscle neurotization in the rat hindlimb model, can lead to good results in terms of skeletal muscle reinnervation. Here we show that, in the rat forelimb model, the combined use of direct muscle neurotization with either end-to-end or end-to-side neurorrhaphy to reinnervate the denervated flexor digitorum muscles, leads to muscle atrophy prevention over a long postoperative time lapse (10 months). By contrast, very little motor recovery (in case of end-to-end neurorrhaphy) and almost no motor recovery (in case of end-to-side neurorrhaphy) were observed in the grasping activity controlled by flexor digitorum muscles. It can thus be concluded that, at least in the rat, direct muscle neurotization after both end-to-end and end-to-side neurorrhaphy represents a good strategy for preventing denervation-related muscle atrophy but not for regaining the lost motor function.

Chapter 1: Peripheral nerve repair and regeneration research: a historical note.

Although the most significant advances in nerve repair and regeneration have been acquired over the last few decades, the study of nerve repair and regeneration potential dates back to ancient times namely to Galen in the second century A.D. This brief historical note outlines the milestones which have guided us to our present knowledge. In particular, we focus on the nineteenth century and the first decades of the twentieth century, an age in which the fathers of neurosurgery and neurobiology established the basis for most of the nerve repair and regeneration concepts used today. Finally, we shine a light on the most current history to show how recent pressure to use modern interdisciplinary and translational approach represents a sort of rediscovery of the scientific habits of the fathers of modern biomedicine, who used to carry out research from an integrated and broad point of view rather than from a super-specialized and specific one as it is often used today.

Chapter 4: Methods and protocols in peripheral nerve regeneration experimental research: part I-experimental models.

This paper addresses several basic issues that are important for the experimental model design to investigate peripheral nerve regeneration. First, the importance of carrying out adequate preliminary in vitro investigation is emphasized in light of the ethical issues and with particular emphasis on the concept of the Three Rs (Replacement, Reduction, and Refinement) for limiting in vivo animal studies. Second, the various options for the selection of the animal species for nerve regeneration research are reviewed. Third, the two main experimental paradigms of nerve lesion (axonotmesis vs. neurotmesis followed by microsurgical reconstruction) are critically outlined and compared. Fourth, the various nerve models that have most commonly been employed are overviewed focusing in particular on forearm mixed nerves and on behavioural tests for assessing their function: the ulnar test and the grasping test which is useful for assessing both median and radial nerves in the rat. Finally, the importance of considering the influence of various factors and diseases which could interfere with the nerve regeneration process is emphasized in the perspective of a wider adoption of experimental models which more closely mimic the environmental and clinical conditions found in patients.

Chapter 14: End-to-side nerve regeneration: from the laboratory bench to clinical applications.

Translation of laboratory results to the patient is a critical step in biomedical research and sometimes promising basic science and preclinical results fail to meet the expectations when translated to the clinics. End-to-side (ETS) nerve regeneration is an example of an innovative neurobiological concept, which, after having generated great expectations in experimental and preclinical studies, provided very conflicting results when applied to clinical case series. A number of basic science studies have shown that ETS neurorrhaphy, in fact, is able to induce collateral sprouting from donor nerve's axons, allowing for massive repopulation of the distal nerve stump. Experimental studies have also shown that ETS neurorrhaphy can recover voluntary control of skeletal muscles and that voluntary motor function recovery can be achieved both with agonistic and antagonistic donor nerves, thus widening the potential clinical indications. However, clinical case series reported so far, did not meet these promises and results have been rather conflicting, especially regarding repair of proximally located mixed nerves. In contrast, ETS reconstruction of distal sensory nerve lesions led to a more positive outcome and, most importantly, consistent results among international centers carrying out clinical trials. Concluding, ETS is a promising microsurgical approach for nerve coaptation, based on a convincing and innovative neurobiological concept. However, conflicting clinical results and disagreement among surgeons regarding its employment suggest that this technique should still be considered an ultima ratio, reserved for cases where no other repair technique can be attempted. New data coming from neurobiological research will help further enlarge the clinical indications of ETS nerve reconstruction, explain the different results found in laboratory animals and humans, and contribute to new treatments and rehabilitation strategies aimed at improving the efficacy of nerve regeneration after ETS neurorrhaphy.

The use of sentinel node biopsy and selective lymphadenectomy in squamous cell carcinoma of the upper limb. Usefulness of sentinel node biopsy to avoid useless lymphadenectomies in high-risk upper limb SCC.

BACKGROUND: Squamous cell carcinoma (SCC) is the second most common skin cancer in humans. Because the incidence of metastasis from SCC of the skin is rare, regional lymphadenectomy is generally not recommended for the patients with clinically node-negative disease. However, in patients with an intermediate and high risk of metastasis, evaluation of the lymph nodes to detect the absence of metastatic nodal disease is a difficult task. PATIENTS AND METHODS: The authors reviewed the pertinent demographic and surgical data in a consecutive series of six patients with squamous cell carcinoma who underwent sentinel lymph node staging. The tumour size was greater than 2 cm (T2) and the patients had clinically non-palpable regional lymph nodes (N0). All nodes were examined using haematoxylin-eosin staining. Sentinel Lymph Node Biopsy (SLNB) and Selective Lymphadenectomy (SL) using preoperative lymphoscintigraphy and intraoperative radiolymphoscintigraphy and vital dye injections was used to identify the sentinel lymph node avoiding complete axillary node dissection. RESULTS: No false-negative results were observed. At a median follow-up of 10 months (mean 15 months), neither local or regional recurrences in sentinel node-negative basins have been noted. CONCLUSIONS: Sentinel node biopsy is a minimally invasive staging procedure useful in identifying occult regional lymph node disease in selected patients with squamous cutaneous malignancies of the arm. Furthermore sentinel lymph node histology is possibly the most important negative predictor of early recurrence and survival in patients with American Joint Committee on Cancer stage I and II squamous cell carcinoma. Although sentinel node-negative patients are a prognostically favourable group, this small series of patients demonstrates that further studies to verify these findings and develop formal guidelines are indicated

Selection of the donor nerve for end-to-side neurorrhaphy.

OBJECT: The authors of other studies have reported that the selection of an agonistic donor nerve is required for recovering voluntary motor control after end-to-side nerve repair. In this experimental investigation, the authors' goal was to verify this assumption by performing end-to-side neurorrhaphy of the rat median nerve on its antagonistic radial nerve. METHODS: The left median nerve in 10 adult female rats was repaired by end-to-side neurorrhaphy after epineuriotomy on the radial nerve at the middle of the brachium. The time course of median nerve functional recovery was then assessed using the grasping test until postoperative Week 30. Before removing the nerve, the surgical site was carefully explored to exclude contamination by the proximal nerve stump, and the functional anatomy of median and radial nerves was assessed by electrical stimulation. Repaired nerves were then processed for resin embedding, and semithin sections were obtained for nerve fiber histomorphometry by using the dissector method. RESULTS: Repaired median nerves were repopulated by nerve fibers regenerating from the radial donor nerve as previously shown. Moreover, voluntary motor control of the flexor muscles innervated by the median nerve was progressively recovered beginning in postoperative Week 10 and reaching 42% of normal by Week 30. CONCLUSIONS: Contrary to previously reported data, recovery of voluntary motor function after end-to-side nerve repair can also be expected when an antagonistic nerve is used as a donor nerve.

The superior pedicle mammaplasty for the treatment of pedunculous breast.

AIM OF THE STUDY: The superior pedicle mammaplasty is a technique frequently employed in the treatment of breast ptosis, associated or not with hypertrophy of the gland, followed by satisfactory results. This technique is normally not indicated in severe breast ptosis (pendulous breast) because of the excessive length of the pedicle supplying nipple-areola-complex (NAC), with the risk of ischemia. In these cases the standard technique is the free-nipple-graft mammaplasty. However, the deepen knowledge about vascular anatomy of the breast and the aptitude to perform superior pedicle mammaplasty, induced the authors to indicate this technique even in these cases improving the aesthetic and functional outcomes. MATERIAL AND METHODS: The authors present a series of 30 patient with pendulous breasts, with sternal notch-nipple distance equal or superior to 32 cm (45 cm maximum; mean value 35.1), treated with the superior pedicle mammaplasty with inverted "T" scar. The results confirm the reliability of superior pedicle for the nipple-areolar complex blood supply, associated with satisfactory aesthetic results due especially to the good breast projection. CONCLUSIONS: They conclude that superior pedicle technique mammaplasty, even if normally not indicated in these cases, is instead suitable for the treatment of pendulous breasts with great sternal notch-nipple distance, permitting to take advantages of this technique.

End-to-side (terminolateral) nerve regeneration: a challenge for neuroscientists coming from an intriguing nerve repair concept.

The last 15 years have seen a growing interest regarding a technique for nerve repair named end-to-side (terminolateral) neurorrhaphy. This technique is based on the concept that nerve fiber regeneration along the distal stump of a transected nerve, the proximal stump of which was lost, can be obtained by just suturing the proximal end of its distal stump to the epinerium of a neighbor healthy and undamaged donor nerve. A large body of experimental studies have shown that end-to-side neurorrhaphy, in fact, is able to induce collateral sprouting from donor nerve's axons which is at the basis of the massive repopulation of the distal nerve stump. The regenerating nerve fibers eventually reinnervate the periphery of the severed nerve leading to a recovery of the lost function the degree of which varies depending on factors that still have to be elucidated. Surprisingly, this puzzling concept of nerve regeneration has attracted very little attention from basic neuroscientists so far and, thus, the present paper is intended to call for more biological research on it by overviewing the relevant literature and indicating the several unanswered questions that this concept asks to the neuroscience community.

The ulnar test: a method for the quantitative functional assessment of posttraumatic ulnar nerve recovery in the rat.

The assessment of recovery of the neuromuscular function following nerve lesion and repair is one of the main goals of peripheral nerve researchers. The forelimb model has recently seen an increase in its employment for experimental nerve repair studies especially because of the availability of the grasping test for assessing the functional recovery of one of its major nerves, the median nerve. Nerve repair studies sometimes require the use of more than one nerve to simulate severe clinical situations and, in this case, the ulnar nerve is often used together with the median nerve. However, a test for assessing ulnar nerve functional recovery is yet not available. To fill this gap, we have developed and experimentally tested a method for the functional assessment of posttraumatic ulnar nerve recovery in the rat. Animal testing using this method is simple, quick and provides the animal with minimal distress. The method proved to be effective in detecting the date on which recovery starts after ulnar nerve impairment and in following its improvement, over time. The availability of this new test is expected to further increase the employment of forelimb experimental nerve models instead of the more disabling hindlimb models.

Morphologic and functional study of rat median nerve repair by terminolateral neurorrhaphy of the ulnar nerve.

While it has been shown that terminolateral (end-to-side) neurorrhaphy leads to successful functional motor reinnervation of the peripheral territories belonging to the severed nerve, data on the morphology of terminolateral sprouting and on the voluntary control of the motor function restored by terminolateral neurorrhaphy are still partial. In this study, the severed rat median nerve was sutured in an end-to-side fashion to the intact ulnar nerve. The progression of recovery of the flexion of the fingers was assessed by means of the grasping test. Seven months after surgery, the rats were sacrificed, and morphologic and morphometric analysis was performed on the regenerated median nerve and on the donor ulnar nerve. Results of the functional assessment showed that voluntary motor control of the muscles innervated by the median nerve was partially and progressively recovered by terminolateral neurorrhaphy, with a mean strength in the flexion of the fingers that reached about 20 percent of normal before sacrifice. Morphologic and morphometric analysis showed that nerve-fiber regeneration occurred in all repaired median nerves. Signs of nerve fiber atrophy were detected in the ulnar nerve distal to the point of suture, suggesting the possible occurrence of secondary damage to the donor nerve after terminolateral neurorrhaphy that should be taken into consideration in a clinical perspective.

On the use of the grasping test in the rat median nerve model: a re-appraisal of its efficacy for quantitative assessment of motor function recovery.

The quantitative assessment of motor function is an important requirement for studies on peripheral nerve injury and repair. So far, most studies on peripheral nerves have been performed on the sciatic nerve model using walking track analysis for assessing motor function. Alternatively, the employment of the median nerve model, which allows motor function evaluation by means of a simple behavioural test named grasping test (GT), have been more recently proposed. In this paper, the efficacy of the GT for the quantitative assessment of motor function recovery is re-appraised and a modified device for its carrying out is described. Finally, the rationale for the employment of the median nerve model as an alternative to the sciatic nerve model is critically discussed.