Hisham Fansa

In vivo predegeneration of peripheral nerves: an effective technique to obtain activated Schwann cells for nerve conduits.

In vivo predegeneration of peripheral nerves is presented as a convenient and effective method to obtain activated Schwann cells and an enhanced cell yield following in vitro cultivation. The experiments conducted in rats were aimed at clinical use in gaining Schwann cell suspensions for filling artificial conduits in order to bridge peripheral nerve gaps. The rat sciatic nerve used as a model was transected distally to the spinal ganglia. Predegeneration in vivo was allowed to take place for 1, 2, 3 and 4 days and up to 1, 2 and 3 weeks. The nerve was then resected and prepared for cell cultivation. Schwann cells cultivated from the contralateral untreated nerve served as control. Immunostaining for S100, nerve growth factor receptor and the adhesion molecules N-cadherin and L1 was used to characterize the general state of the cultures. Viability was assessed by fluorescein fluorescence staining, and the proliferation index was determined by bromodeoxyuridine-DNA incorporation. The Schwann cells from predegenerated nerves revealed an increased proliferation rate compared to the control, whereas fibroblast contamination was decreased. Best results were obtained 1 week after predegeneration.

Magnetic nanoparticles in primary neural cell cultures are mainly taken up by microglia

BackgroundMagnetic nanoparticles (MNPs) offer a large range of applications in life sciences. Applications in neurosciences are one focus of interest. Unfortunately, not all groups have access to nanoparticles or the possibility to develop and produce them for their applications. Hence, they have to focus on commercially available particles. Little is known about the uptake of nanoparticles in primary cells. Previously studies mostly reported cellular uptake in cell lines. Here we present a systematic study on the uptake of magnetic nanoparticles (MNPs) by primary cells of the nervous system.ResultsWe assessed the internalization in different cell types with confocal and electron microscopy. The analysis confirmed the uptake of MNPs in the cells, probably with endocytotic mechanisms. Furthermore, we compared the uptake in PC12 cells, a rat pheochromocytoma cell line, which is often used as a neuronal cell model, with primary neuronal cells. It was found that the percentage of PC12 cells loaded with MNPs was significantly higher than for neurons. Uptake studies in primary mixed neuronal/glial cultures revealed predominant uptake of MNPs by microglia and an increase in their number. The number of astroglia and oligodendroglia which incorporated MNPs was lower and stable. Primary mixed Schwann cell/fibroblast cultures showed similar MNP uptake of both cell types, but the Schwann cell number decreased after MNP incubation. Organotypic co-cultures of spinal cord slices and peripheral nerve grafts resembled the results of the dispersed primary cell cultures.ConclusionsThe commercial MNPs used activated microglial phagocytosis in both disperse and organotypic culture systems. It can be assumed that in vivo application would induce immune system reactivity, too. Because of this, their usefulness for in vivo neuroscientific implementations can be questioned. Future studies will need to overcome this issue with the use of cell-specific targeting strategies. Additionally, we found that PC12 cells took up significantly more MNPs than primary neurons. This difference indicates that PC12 cells are not a suitable model for natural neuronal uptake of nanoparticles and qualify previous results in PC12 cells.

Differences in peripheral nerve degeneration/regeneration between wild-type and neuronal nitric oxide synthase knockout mice.

Nitric oxide (NO), a unique biological messenger molecule, is synthesized by three isoforms of the enzyme NO synthase (NOS) and diffuses from the site of production across cellular membranes. A postulated role for NO in degeneration and regeneration of peripheral nerves has been explored in a sciatic nerve model comparing wild‐type mice and mice lacking neuronal NOS after transection and microsurgical repair. In NOS knockout mice, regenerative delay was observed, preceded by a decelerated Wallerian degeneration (WD). In the regenerated nerve, pruning of uncontrolled sprouts was disturbed, leading to an enhanced number of axons, whereas remyelination seemed to be less affected. Delayed regeneration was associated with a delayed recovery of sensor and motor function. In such a context, possible NO targets are neurofilaments and myelin sheaths of the interrupted axon, filopodia of the growth cone, newly formed neuromuscular endplates, and Schwann cells in the distal nerve stump. The results presented suggest that 1) local release of NO following peripheral nerve injury is a crucial factor in degeneration/regeneration, 2) success of fiber regeneration in the peripheral nervous system depends on a regular WD, and 3) manipulation of NO supply may offer interesting therapeutic options for treatment of peripheral nerve lesions.

Neuronal nitric oxide synthase is the dominant nitric oxide supplier for the survival of dorsal root ganglia after peripheral nerve axotomy

This study was designed to determine whether nitric oxide supply may be a major factor in the survival of dorsal root ganglia in a sciatic nerve injury model. Wild-type (WT) mice were compared with knockout (KO) mice lacking neuronal nitric oxide synthase (nNOS) or endothelial (eNOS). The NO-generating capacities were analysed by NOS immunohistochemistry and NADPH-diaphorase staining 1, 2, 6, and 12 weeks after nerve transection. The occurrence and morphological type of neuronal death were determined by TUNEL reaction and ultrastructural examination. Cell loss following nerve section, whist dependent on the availability of NO, as shown by its marked elevation in nNOS KO mice, did not correlate well with nNOS expression in WT animals. Whereas a lack of eNOS was tolerated, deficiency of nNOS led to an enhanced cell loss. The results suggest a crucial role of NO supply after transection of peripheral nerves with a particular significance of the nNOS isoform.

The Transverse Myocutaneous Gracilis Muscle Flap: A Fast and Reliable Method for Breast Reconstruction

Background: Autologous breast reconstruction is predominantly performed using free transverse rectus abdominis musculocutaneous or deep inferior epigastric perforator flaps. However, some patients are not suitable candidates for flaps from the lower abdomen. The transverse skin island of the gracilis muscle presents an additional option, as it includes tissue from the posterior upper thigh/lower buttock and thus delivers the amount of tissue necessary for breast reconstruction. Methods: In 2007, the authors’ unit performed 73 free flaps for breast reconstruction subsequent to carcinoma, implant-related capsular fibrosis, and breast asymmetry. The transverse myocutaneous gracilis flap was used 32 times. The ventral margin was the greater saphenous vein, and the posterior margin was the midline of the inferior gluteal fold. The skin island could be harvested to a width of up to 30 cm and a height of up to 10 cm. The donor site was closed primarily. Results: Thirty-two flaps were performed in 20 patients. Mean follow-up was 6 months. Mean operating time was 220 minutes for unilateral and 325 minutes for bilateral cases. All flaps and donor sites healed uneventfully. An initially described “tight feeling” at the thigh ceased after 2 to 3 weeks. Persistent hypesthesia of the dorsal thigh was not noted. A major asymmetry of the thigh in unilateral transplantations was not apparent. After 6 months, all flaps were soft. Conclusions: The transverse myocutaneous gracilis flap is a safe, fast flap for reconstruction after benign and malignant breast disease. It combines a constant vascular pedicle with soft subcutaneous tissue that has breast-like characteristics.

Influence of insulin‐like growth factor‐I (IGF‐I) on nerve autografts and tissue‐engineered nerve grafts

To overcome the problems of limited donor nerves for nerve reconstruction, we established nerve grafts made from cultured Schwann cells and basal lamina from acellular muscle and used them to bridge a 2‐cm defect of the rat sciatic nerve. Due to their basal lamina and to viable Schwann cells, these grafts allow regeneration that is comparable to autologous nerve grafts. In order to enhance regeneration, insulin‐like growth factor (IGF‐I) was locally applied via osmotic pumps. Autologous nerve grafts with and without IGF‐I served as controls. Muscle weight ratio was significantly increased in the autograft group treated with IGF‐I compared to the group with no treatment; no effect was evident in the tissue‐engineered grafts. Autografts with IGF‐I application revealed a significantly increased axon count and an improved g‐ratio as indicator for “maturity” of axons compared to autografts without IGF‐I. IGF‐I application to the engineered grafts resulted in a decreased axon count compared to grafts without IGF‐I. The g‐ratio, however, revealed no significant difference between the groups. Local administration of IGF‐I improves axonal regeneration in regular nerve grafts, but not in tissue‐engineered grafts. Seemingly, in these grafts the interactive feedback mechanisms of neuron, glial cell, and extracellular matrix are not established, and IGF‐I cannot exert its action as a pleiotrophic signal.

Host responses after acellular muscle basal lamina allografting used as a matrix for tissue engineered nerve grafts1.

Background. A nerve gap must be bridged by autologous nerve grafts that serve as scaffold and consist of viable Schwann cells that promote regeneration. Owing to the necessary immunosuppression, nerve allografts remain limited to special cases. Alternatively, tissue engineering of peripheral nerves focuses on the implantation of cultured Schwann cells into suitable scaffolds. We established grafts from Schwann cells and basal lamina from acellular muscles. These grafts offer a regeneration that is comparable to autologous nerve grafts. Methods. Using a rat model (DALEW.1W strain), the present study evaluates the host response to acellular muscle allografts by assessing cellular reaction major histocompatability (MHC) class I and II, lymphocytes, macrophages. The results were compared to untreated muscle allografts. Results. Macroscopically, the untreated muscles showed a strong inflammatory reaction as a sign of rejection, whereas the acellular muscle offered only minor reactions in the periphery of the graft. Expression of MHC I and II and invasion of CD4/CD8 positive cells and macrophages was pronounced after grafting the untreated muscles. Only a moderate reaction was noted for these parameters after acellular grafting. Conclusions. The acellular muscle graft is not completely free of cellular response; however the reaction is considered to be moderate and is located only in the periphery. To date, synthetic scaffolds that represent endoneurial tube-like structures and allow sufficient adhesion of Schwann cells and axonal regeneration are not available. The decreased response to acellular muscle allografts offers at least a basis for further experiments.

Biocompatibility of Different Nerve Tubes

Bridging nerve gaps with suitable grafts is a major clinical problem. The autologous nerve graft is considered to be the gold standard, providing the best functional results; however, donor site morbidity is still a major disadvantage. Various attempts have been made to overcome the problems of autologous nerve grafts with artificial nerve tubes, which are “ready-to-use” in almost every situation. A wide range of materials have been used in animal models but only few have been applied to date clinically, where biocompatibility is an inevitable prerequisite. This review gives an idea about artificial nerve tubes with special focus on their biocompatibility in animals and humans.

Revascularization of tissue-engineered nerve grafts and invasion of macrophages.

Nonneural derived nerve conduits fail to support regeneration over larger gaps due to lacking viable Schwann cells. Thus, tissue engineering of nerves is focusing on implantation of viable Schwann cells into suitable scaffolds. We established grafts made from acellular muscles and veins, respectively, seeded with cultured Schwann cells. As timing of revascularization is crucial to determine Schwann cell survival and depending axonal regeneration we studied establishment of vascular architecture in a rat sciatic nerve model (2-cm gap) after 3, 5, 7, and 10 days postoperatively, using albumin bound Evans blue. Additionally, macrophage recruitment was immunohistochemically assessed. Engineered grafts showed a delayed revascularization, starting between day 5 and 7 in comparison to normal autografts, that revascularized by day 3. Macrophage recruitment in autologous nerve grafts was evident by day 3. The engineered groups revealed no macrophage invasion until day 7. As Schwann cells survive up to 7 days in autologous grafts without blood supply, depending purely on diffusion, establishment of vascular structure between day 5 and 7 is rapid enough to support Schwann cell survival in engineered grafts. As these grafts are lacking Wallerian degeneration delayed macrophage invasion may not impair degeneration-dependent regeneration, but presence of macrophage derived or induced growth factors may be decreased.

Minocycline protects Schwann cells from ischemia-like injury and promotes axonal outgrowth in bioartificial nerve grafts lacking Wallerian degeneration

Minocycline, a broad-spectrum antimicrobial tetracycline, acts neuroprotectively in ischemia. Recently, however, minocycline has been revealed to have ambiguous effects on nerve regeneration. Thus its effects in a rat sciatic nerve transplantation model and on cultivated Schwann cells stressed by oxygen glucose deprivation (OGD) were studied. The negative effect of minocycline on Wallerian degeneration, the essential initial phase of degeneration/regeneration after nerve injury, that was recently demonstrated, was excluded by using predegenerated nerve and Schwann cell-enriched muscle grafts, both free of Wallerian degeneration. They were compared with common nerve grafts. The principle findings were that in vitro minocycline provided protective effects against OGD-induced death of Schwann cells by preventing permeability of the mitochondrial membrane. It suppressed the OGD-mediated induction of HIF-1alpha and BAX, and stabilized/induced BCL-2. Cytochrome c release and cleavage of procaspase-3 were diminished; release and translocation of AIF and cytotoxic cleavage of actin into fractin were stopped. In common nerve grafts, minocycline, besides its direct anti-ischemic effect, hampered revascularization by down-regulation of MMP9 and VEGF prolonging ischemia and impeding macrophage recruitment. In bioartificial nerve grafts that were free of Wallerian degeneration and revealed lower immunogenicity, minocycline aided the regeneration process. Here, the direct anti-ischemic effect of minocycline on Schwann cells, which are mandatory for successful peripheral nerve regeneration, dominated the systemic anti-angiogenic/pro-ischemic effects. In common nerve grafts, however, where Wallerian degeneration is a prerequisite, the anti-angiogenic and macrophage-depressing effect is an obstacle for regeneration.