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Most tumours are derived from a single cell that is transformed into a cancer-initiating cell (cancer stem cell) that has the capacity to proliferate and form tumours in vivo. However, the origin of the cancer stem cell remains elusive. Interestingly, during development and tissue repair the fusion of genetic and cytoplasmic material between cells of different origins is an important physiological process. Such cell fusion and horizontal gene-transfer events have also been linked to several fundamental features of cancer and could be important in the development of the cancer stem cell.

In glioma cells, the stimulatory input of extracellular matrix components and an increased sensitivity to growth factors result in a high proliferative and migratory behaviour. Cell surface receptor interactions play pivotal roles in converging information about conditions in the environment immediately outside the cell. The transduced signal, in turn induces a response within the cell that provokes a specific behaviour. Cellular migration and cell proliferation are interwoven processes that share several common intracellular pathways. The major cross-links are the phosphoinositol phosphate regulating enzymes, PI-3 kinase and PTEN, the focal adhesion kinase (FAK) and the tumour suppressor p53. An understanding of the interaction between the molecular participants involved in migration and proliferation will promote the design of new treatments. A full understanding of the basis of the invasiveness of tumour cells remains elusive. Gene and protein expression are being studied, using modern techniques such as microarray analysis, SAGE and 2-D protein gels. Transient and permanent protein-protein interactions and recruitment of proteins to specialised cellular domains are equally important in regulating cellular invasion and presumably will attract more attention in future.

An anesthesiological management is difficult in surgical procedures in which the operative situs is not to be seen by the anesthesist. Therefore specific knowledge of the operative procedure and related hazards are mandatory for optimal anesthesiological care. By describing the anesthesiologic proceduces in two lumbar discectomys, the specific problems in recognition and treatment of severe injuries of retroperitoneal vascular structures are explained. In addition, the differential diagnosis of intraoperative arterial hypotension is described. The communication between all disciplines involved is mandatory, especially in cases of severe complications. The management of such complications can only be solved through in-time interdisciplinary cooperation of all involved disciplines.

Paclitaxel (Taxol), an anti-cancer drug derived from Taxus species, was tested for its anti-migrational, anti-invasive and anti-proliferative effect on two human glioma cell lines (GaMg and D-54Mg) grown as multicellular tumour spheroids. In addition, the direct effect of paclitaxel on glioma cells was studied using flow cytometry and scanning confocal microscopy. Both cell lines showed a dose-dependent growth and migratory response to paclitaxel. The GaMg cells were found to be 5-10 times more sensitive to paclitaxel than D-54Mg cells. Paclitaxel also proved to be remarkably effective in preventing invasion in a co-culture system in which tumour spheroids were confronted with fetal rat brain cell aggregates. Control experiments with Cremophor EL (the solvent of paclitaxel for clinical use) in this study showed no effect on tumour cell migration, cell proliferation or cell invasion. Scanning confocal microscopy of both cell lines showed an extensive random organization of the microtubules in the cytoplasm. After paclitaxel exposure, the GaMg and the D-54Mg cells exhibited a fragmentation of the nuclear material, indicating a possible induction of apoptosis. In line with this, flow cytometric DNA histograms showed an accumulation of cells in the G2/M phase of the cell cycle after 24 h of paclitaxel exposure. After 48 h, a deterioration of the DNA histograms was observed indicating nuclear fragmentation. Images Figure 3 Figure 6

Fetal as well as mature neural cells were homografted into the right cerebral hemisphere of adult BD-IX rats. The animals were sacrificed 7 d after implantation, and the localization of implanted cells was visualized by fluorescence and light microscopy. The cell implants were prestained with the fluorescent vital dye 1,1'-Dioctadecyl-3,3,3'3'-tetramethylindocarbocyanine perchlorate (DiI) to discriminate between implanted cells and host brain tissue. At the implantation site, the fetal brain cells as well as the cells from immature brain cell aggregates showed diffuse infiltration into the surrounding host brain tissue of up to 0.5 mm. Extensive cell migration along the corpus callosum for up to 5 mm in the coronal and to a lesser extent in the sagittal plane was also observed. In addition, fetal cells were distributed in the subarachnoid space of both cerebral hemispheres and showed a distinct association with larger blood vessels. Cells from mature brain aggregates did not migrate as far as fetal cells and showed only a local infiltration into the host neuropil. Fluorescent microspheres as well as fixed fetal brain cells were implanted, either alone or in combination with vital cells to distinguish between active cell migration and passive cell displacement. The microspheres and the fixed cells were found either localized to the implantation pathway or distributed in the corpus callosum for up to 2 mm in the coronal plane without any dispersion in the sagittal plane. The microspheres also showed an extensive displacement in the subarachnoid space. In vitro co-culture experiments between two immature aggregates showed a complete fusion of the two aggregates during a 96 h culture period. In co-cultures between two mature aggregates complete fusion was not prominent, although the confrontation zone appeared diffuse. Confrontations between a mature and an immature aggregate showed the same pattern of interaction as seen for the two mature aggregates. It is concluded that carbocyanine dyes may be used as a tracer for transplanted cells. Cells from fetal rat brain cell aggregates, opposed to those from mature aggregates, showed extensive migration along well defined anatomical structures in the mature along well defined anatomical structures in the mature brain. Some of the spread of cells following implantation is probably due to passive movement since inert microspheres will spread into certain areas of the CNS.

The present study describes the effects of 2 folate antagonists, methotrexate (MTX) and the lipophilic antifolate trimetrexate (TMX) on 2 permanent human glioma cell lines (GaMg and D-54Mg) grown as monolayers and as multicellular tumor spheroids. In addition, the effects of drug exposure on tumor cell invasion was studied using a three-dimensional organ co-culture system. In monolayer cultures, TMX was a more potent inhibitor of cell growth than MTX, especially towards the GaMg cell line. The 2 drugs, however, showed similar cytotoxicity as assessed by the plating efficiency assay. Reduced ability of directional migration of cells on a plastic surface was seen by either antifolate usually at concentrations to 10-fold higher than those exerting a cytotoxic effect in the plating efficiency assay. TMX was somewhat more potent than MTX as an inhibitor of spheroid growth. When tumor spheroids were exposed to MTX or TMX at concentrations that caused 65 to 70% inhibition of cell migration, there was a latent period of 4 to 5 days before inhibition of spheroid growth ensued. Invasion was investigated in a co-culture system, where tumor spheroids were confronted with fetal rat brain cell aggregates. Neither drug reduced tumor cell invasion, although histological examination revealed toxic effects both in GaMg and in D-54Mg spheroids. We conclude that spheroids from human glioma cells were less sensitive to the antifolates than monolayers. For both drugs a latency period was observed before inhibition of spheroid growth. The spheroids retained their ability to invade normal brain tissue when exposed to levels of folate antagonists inhibiting spheroid growth.

Previous work concerning the efficacy of different types of nerve repair has been reviewed. We describe a model in which epineurial and perineurial suture techniques were studied by quantitative ultramicroscopy and electrophysiology. In this model, we believe suture of the perineurium was shown to have a slight advantage over suture of the epineurium. Microscopic control and fascicular alignment are advocated to increase the percentage of axons growing into the distal stump and the periphery. The model described should lend itself to the study of other traumatic peripheral neuropathies.

Concerning hyperthermia treatment, knowledge of time-temperature and of temperature distributions within tumour volumes is essential in order to obtain the maximal therapeutic effect. New techniques are developed to overcome these difficulties. Two different heat sources, the contact Nd:YAG laser system and the automatically controlled high frequency current are investigated. In a defined volume of 1 cm3, the laser system reaches 45 degrees C after 1.8 s exposure with 15 W output power. The high frequency current reaches 45 degrees C with 48 s exposition with 18.75 W output current. Both heat sources present an exponential decrease of the temperature profile depending on the distance and prove efficient for inducing anti-tumoural hyperthermia. The tissue heat clearance is compensated for by intermittent laser and high frequency current application.

By serial transplantation of human glioblastoma biopsies into the brain of immunodeficient nude rats, two different tumor phenotypes were obtained. Initially, the transplanted xenografts displayed a highly invasive phenotype that showed no signs of angiogenesis. By serial transplantation in animals, the tumors changed to a less invasive, predominantly angiogenic phenotype. To identify novel proteins related to the invasive phenotype, the xenografts were analyzed using a global proteomics approach. One of the identified proteins was protein disulfide isomerase (PDI) A6 precursor. PDI is a chaperone protein that mediates integrin-dependent cell adhesion. It is both present in the cytosol and at the cell surface. We show that PDI is strongly expressed on invasive glioma cells, in both xenografts and at the invasive front of human glioblastomas. Using an in vitro migration assay, we also show that PDI is expressed on migrating glioma cells. To determine the functional significance of PDI in cell migration, we tested the effect of a PDI inhibitor, bacitracin, and a PDI monoclonal antibody on glioma cell migration and invasion in vitro. Both tumor spheroids derived from human glioblastoma xenografts in nude rat brain and cell line spheroids were used. The PDI antibody, as well as bacitracin, inhibited tumor cell migration and invasion. The anti-invasive effect of bacitracin was reversible after withdrawal of the inhibitor, indicating a specific, nontoxic effect. In conclusion, using a global proteomics approach, PDI was identified to play an important role in glioma cell invasion, and its action was effectively inhibited by bacitracin. (Cancer Res 2006; 66(20): 9895-902)

Most tumours are derived from a single cell that is transformed into a cancer-initiating cell (cancer stem cell) that has the capacity to proliferate and form tumours in vivo. However, the origin of the cancer stem cell remains elusive. Interestingly, during development and tissue repair the fusion of genetic and cytoplasmic material between cells of different origins is an important physiological process. Such cell fusion and horizontal gene-transfer events have also been linked to several fundamental features of cancer and could be important in the development of the cancer stem cell.

The potential benefit of continuous local administration of antiangiogenic proteins to CNS tumors in vivo has recently been demonstrated using endostatin-producing recombinant cells encapsulated in alginate beads. Due to the treatment potential of transplanted alginate-encapsulated cells producing therapeutic proteins, we describe a successful method of cryopreservation (CP) of such beads, in which cellular viability, alginate structure, and protein secretion were maintained. Alginate beads containing human embryonic kidney cells (HEK 293 cells) stably transfected with the gene encoding for endostatin were cryopreserved in dimethyl sulfoxide (DMSO) using a slow freezing procedure. Briefly, the DMSO concentration was gradually increased prior to the freezing procedure. The cryotubes were further supercooled to −7.5°C and nucleated. Thereafter, the samples were cooled at a rate of 0.25°C/min and stored in liquid nitrogen. The viability of the encapsulated cells was assessed using confocal microscopy quantification (CLSM) technique and a MTS assay. The cell cycle distribution inside the beads was assessed by DNA flow cytometry and endostatin production was determined by an endostatin-specific ELISA assay, both prior to and after CP. CLSM measurements showed sustained esterase activity in the beads after thawing, with only a slight transient decrease 24 h after CP. The MTS assay verified these findings by displaying similar variations of intracellular dehydrogenase activity. Flow cytometric analyses revealed no cryorelated disturbances in cellular ploidy. Furthermore, ELISA measurements showed a well-preserved endostatin production after CP. In conclusion, this work describes the successful CP of alginate-encapsulated recombinant cells secreting a therapeutic protein. Together with previous published reports, these results further substantiate the feasibility and potential of cell encapsulation therapy in the treatment of malignant tumors.

Temozolomide is an alkylating cytostatic drug that finds increasing application in the treatment of melanoma, anaplastic astrocytoma and glioblastoma multiforme. The compound is a prodrug that decomposes spontaneously, independent of an enzymatic activation step. DNA methylation induces futile mismatch repair cycles and depletion of the DNA repair enzyme O(6)-methylguanine-DNA methyltransferase should then initiate programmed cell death. We show drug-dependent inhibition of tumour growth in a three-dimensional cell culture model of the glioma cell lines U87MG and GaMG. Migrational behaviour of the glioblastoma cells remained unaltered. However, coincubation of tumour spheroids with primary brain aggregates showed reduced tumour cell invasion into brain tissue in the presence of temozolomide. This was not achieved by slowing cellular migration, as temozolomide-treated cells displayed no reduced motility. By transferase-mediated dUTP nick-end labelling (TUNEL) of apoptotic nuclei, we found that the drug was able to induce apoptosis throughout the tumour cell spheroids. Apoptosis was highest in the core region of the spheroids. Repetitive application of sublethal doses of temozolomide to multicellular spheroids resulted in the development of drug resistance in GaMG cells. We suggest that temozolomide is a strong initiator of apoptosis in glioblastoma tumour cells in a spheroid cell culture system, when cells are already in a stressful environment.

OBPP is a disease with deleterious medical, psychological, and socioeconomic sequelae for the patient and his or her family. The majority of patients show acceptable spontaneous recovery with nonoperative management, which includes aggressive physical rehabilitation and electrical stimulation. There are patients with guarded prognosis, however, who have no chance of recovery unless they undergo early aggressive surgical reconstruction of the injured brachial plexus. It is of great importance to diagnose the injury type as early as possible, especially if there is minimal recovery in the early weeks of follow-up. This allows timely reconstruction to take place, earlier than 3 months for global palsy cases and at 3 months in Erb's type lesions, for best functional results.

The attempts to reconstruct a transected trunk nerve with a free nerve graft of equal diameter have been hindered by the problem of delayed establishment of blood supply to the nerve graft and development eventually of central necrosis and finally failure of nerve regeneration. The transfer of a nerve graft which maintains its blood supply through its vascular pedicle that is microsurgically anastomosed to the recipient site vessels, provided the reconstructive microsurgeon with a new perspective to the clinical problem. A vascularized nerve graft diminishes endoneurial scarring by maintaining the Schwann cell population and decreasing fibroblast infiltration providing, thus, an optimal nutritional environment that results in an increased rate of axonal regeneration.

Treatment of the short nerve gap remains a challenge for the reconstructive surgeon, but it is a clinical problem that can be addressed by nerve expansion. In the present study, the effects of slow nerve expansion on the walking behavior of the rat were examined. When expansion was applied on a normal sciatic nerve or on a transected nerve at either the proximal or the distal segments, permanent 30% elongation could be achieved. The recovered function from the expanded nerve stumps was compared with such classical methods of nerve reconstruction as nerve graft, coaptation under moderate tension, and tensionless repair. The results compared favorably between the expanded groups and the time-honored methods of nerve repair. Analysis of the behavioral data indicated that any amount of expansion affected the functional capabilities of the involved nerve. However, expansion of a normal nerve and/or proximal segment of a transected nerve was better tolerated than distal segment expansion, which suggests that the presence of an axon may have a beneficial effect in minimizing the deforming mechanical insult. Slow nerve expansion appears to have a definite role in the microsurgical management of the short nerve gap.

During embryonic development a complex interplay takes place between the primitive neural ectoderm and surrounding mesodermal tissues. This process is characterized by generation of neuroblasts from the early neuroepithelium which first migrate and then differentiate into mature neural cells in the various regions of the central nervous system (CNS). Extensive cell migration, as observed for fetal cells during development, is a property which is also observed in malignant brain tumor cells in vivo [2–4, 9, 13, 22]. Glioma cells, for instance, show a tendency to migrate along fiber tracts of the white matter and along blood vessels [2, 9], and it is at present unclear whether this migration is enhanced by specific biological properties of the tumor cells or by specific anatomical structures in the brain itself. Of particular interest is the fact that also fetal brain cells, upon transplantation into the adult brain, show a tendency for extensive migration within the CNS [11, 12, 18]. In addition, it has been shown that tumor cells can possess specific fetal phenotyp-ic properties [14, 19] which may be acquired during the process of transformation and differentiation. In the present study we therefore compared the migratory patterns of fetal brain cells and glioma cells transplanted into the adult CNS. To visualize the transplanted cells within the brain they were stained with the carbocyanine dye 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine Perchlorate (DiI), which is nontoxic and integrates within the plasma membrane of individual living cells [15–17].

To study partial facial palsies, to understand the mechanism of the deformity, and to design therapeutic schemes to reverse the facial imbalance.

To critically analyze microreconstructive schemes aiming at the functional substitution of the paralyzed eye sphincter.

To present the introduction and our 6-year clinical experience of a new technique for facial reanimation by which ipsilateral “strong” motor donors are used to preserve denervated targets in complete or partial facial palsies until contralateral VII nerve motor fibers take over.

This report proposes that reflex sympathetic dystrophy be defined as a pain syndrome in which the pain is accompanied by loss of function and evidence of autonomic dysfunction. In the clinical setting, this diagnosis is usually associated with other anatomic and psychological diagnoses and may be associated with a variety of systemic illnesses and medicolegal factors. All components should be assessed before a treatment plan is established. Priorities should go to emergency care, acute injuries, and systemic illness, psychiatric problems, and chronic anatomic problems, in that order. Early, accurate diagnosis improves prognosis.

The primary aim in reconstructive surgery of the upper extremity is to provide tactile gnosis to sensory depleted areas. It is essential for restoration of function and successful rehabilitation of the injured hand. Neurovascular free flaps may provide the answer to this catastrophic problem and transform a useless appendage into a functional gripping hand. Sensory mapping techniques can greatly assist the surgeon in precisely defining new donor sites for these transfers. Cutaneous sensibility, cutaneous sensation, and hand function are subjects of great complexity. Much more investigative and clinical work is needed in this challenging field to improve our evaluation of the sensory injury to the hand and to provide new avenues for restoring functional sensibility in the upper extremity.

Results with peripheral nerve injuries, which today still fall short of our goals, should intensify our efforts in this specialized field of microsurgery. We are living in an exciting era of functional reconstitution. Long gone are the times when a pink replanted digit or a successful free flap comprised the end-point of our surgical efforts. The direction from now on is toward achieving functional restoration by utilizing basic principles while armed with the unlimited potentials of reconstructive microsurgery. To this end we need the assistance and cooperation of other sciences since the enigma of the peripheral nerve can be deciphered only by a multidisciplinary approach.

The authors' experience in the management of brachial plexus injuries over three periods of evolution is presented. Developments in microsurgery have expanded the strategies in brachial plexus management, and these are discussed in full.

1. A total of 791 fibers were isolated from the ulnar nerves of five baboons. Over half of these were obtained from the right ulnar nerves subsequent to reinnervation following a nerve crush; the other fibers were obtained from the undamaged left ulnar nerves. 2. The conduction velocities in the proximal portion of the injured axons dropped below normal, and this reduction persisted until reinnervation appeared nearly complete. 3. The response properties of 65 cutaneous afferent fibers serving reinnervated glabrous skin were compared to 80 fibers from normal skin. 4. Of the afferent fibers reinnervating skin, the proportion judged to have abnormal response properties was not significantly greater than the proportion in normal skin. 5. After reinnervation, cutaneous rapidly adpating fibers displayed tuning curves characteristic of their submodality, while some cutaneous slowly adapting fibers could still be differentiated into type I and type II fibers. However, both types of slowly adapting fibers displayed an increased rate of adaptation and a lowered sensitivity to sustained displacements even at 5 mo following reinnervation. 6. In conclusion, the cutaneous mechanoreceptors in reinnervated glabrous skin regained response properties that allowed them to be assigned to the same submodalities found in normal skin. Submodality was recognizable at an early stage when the receptive field was still immature and when the threshold was elevated. With time, the receptive-field sizes and shapes returned to normal, the thresholds approached normal, and the remaining differences from normal cutaneous afferent fibers became minor.

One of the great difficulties in the development of surgery of parenchymal organs is the control of bleeding during dissection. This can be accomplished by cutting within intersegmental planes or by temporary occlusion of the vessels. Selective separation of tissue from vessels and fibrous structures is possible by focused ultrasonic energy (e.g., CUSA-System), which has found increasing application in liver and brain surgery. Another possibility of selective dissection is the method of water-jet cutting.

Many large-diameter myelinated axons in spinal dorsal roots contain carbonic anhydrase activity, whereas few small-diameter ventral root axons stain for this enzyme. This differential localization of carbonic anhydrase in sensory and motor nerve fibers is indicative of the potential of carbonic anhydrase histochemistry to provide a convenient method for identifying predominantly motor or sensory fascicles in cut ends of peripheral nerves, thereby facilitating coaptation of fascicles in peripheral nerve repair.

A scheme for surgery in patients with avulsed roots is represented by a rat model whose left sciatic nerve was grafted upon the right sciatic nerve, the proximal end of the graft being introduced into the spinal cord white matter. Transcranial stimulation, EMG recording, HRP and histological studies proved that the communication between the brain and the denervated muscles may be reestablished via the motoneurons, through the graft, bypassing the motor roots. Behavioral techniques were developed and will be tested in the future. The possibility of using neurotrophic drugs will be considered.

The water jet cutting system allows transaction and dissection of biological structures with little bleeding. Structures of higher tissue rigidity remain unchanged while softer tissues are mechanically dissected. In brain tissue, all vessels larger than 20 microns are left intact after the passage of the jet stream with a pressure of up to 5 bar, and therefore vessels can be isolated selectively from the surrounding tissue. Oedema is present adjacent to the cut and no increase of temperature occurs.

Replanted digital survivals range between 85% to over 90% at established microsurgery centers around the world. The time has come that all surgeons engaged in this activity must utilize the basic principles of reconstructive hand surgery in order to achieve the maximum functional return of the replanted parts. The vascular repair is only one step in the road toward functional restoration of the replanted part.

Our presently inadequate knowledge of the functional anatomy of the deep branch of the ulnar nerve handicaps our management of ulnar nerve lesions. The extensive anatomical variations in the distribution of this nerve preclude adherence to a textbook pattern of innervation. Electrophysiological recordings of single sensory fibers in monkeys along with fascicular stimulation studies provided novel information about the functional organization of the deep branch of the ulnar nerve: (1) there is a wide range of sensory fibers not currently recognized by textbook descriptions; (2) the innervation pattern is far more diffuse and covers greater areas than previously suspected; (3) at the distal forearm level the motor component of the deep ulnar branch is distributed throughout the entire ulnar nerve and is not restricted to specific fascicles; (4) contrary to previous assumptions, the deep branch of the ulnar nerve is mixed, and not purely motor. In fact, it may be mostly sensory. Afferent fibers arise from muscle, joints, deep subcutaneous tissues and even skin. These findings suggest that it is unwise to look at any nerve as purely motor or having a set innervation pattern, and emphasize the pressing need for objective preoperative and/or intraoperative functional assessment in peripheral nerve surgery.

This article supplies data related to 11 operated children with obstetric brachial plexus palsy. Discussion of the obstetric background, pathology, pre- and postsurgical testing and of primary and secondary surgical reconstruction also is given. The need for rehabilitation regimen is stressed.

This study has provided baseline data of a quantitative neurophysiological appraisal of normal breast sensibility using our current understanding of cutaneous mechanoreceptive function. Test variables, including pressure, low- and high-frequency vibration, static and moving two-point discrimination, temperature, nipple erectibility, and pain, were evaluated in 11 normal volunteers using different coordinates over the nipple, areola, and body of the breast. Definite patterns were established so that the breast could be subdivided into distinct cutaneous areas with differing mechanoreceptive characteristics. The future aim is to broaden the scope of this study with preoperative evaluations in our clinical patients so that appropriate recommendations for future reconstructive breast procedures can be made.

Nerve grafting has made advances in the last 100 years. One of the most significant of these was realizing the importance of adequate blood supply for successful nerve grafting. The vascular anatomy of the six potential vascularized donor nerves we have reviewed may prove to be extremely useful in reconstructing proximal nerve lesions, large nerve defects, and compromised recipient beds.

The author introduced this muscle for the first time almost a decade ago, and this is the first extensive description of the intricate microanatomy of this complex but unique microneurovascular muscle unit. Advantages and disadvantages and indications and contraindications for its use in facial paralysis are presented in detail from an extensive clinical experience of almost 50 such microneurovascular transfers. Pitfalls that the reconstructive microsurgeon should beware and strengths in using this muscle for facial palsy are highlighted. The detailed operative approach is presented, with promise of undetectable scars and minimal functional loss. The strategies for how to inset this muscle unit in the new recipient site are given, along with the thought processes involved in selecting the actual sites of anchoring the muscle to reproduce a mirror image of the contralateral normal face. Finally, an exemplary clinical case demonstrating the use of the pectoralis minor muscle for both eye and lower face reanimation is presented in detail, demonstrating the dual nerve supply and the resulting independent eye and smile movements with total lack of mass action and/or synkinesis. Restorations of eye blink and of a symmetrical and coordinated smile are the frequent rewards of using this unique muscle for the correction of facial palsy.