Sciatic nerve regeneration is not inhibited by anti-NGF antibody treatment in the adult rat.

Elevated nerve growth factor (NGF) is believed to play a role in many types of pain. An NGF-blocking antibody (muMab 911) has been shown to reduce pain and hyperalgesia in pain models, suggesting a novel therapeutic approach for pain management. Since NGF also plays important roles in peripheral nervous system development and sensory nerve outgrowth, we asked whether anti-NGF antibodies would adversely impact peripheral nerve regeneration. Adult rats underwent a unilateral sciatic nerve crush to transect axons and were subcutaneously dosed weekly for 8weeks with muMab 911 or vehicle beginning 1day prior to injury. Plasma levels of muMab 911 were assessed from blood samples and foot print analysis was used to assess functional recovery. At 8-weeks post-nerve injury, sciatic nerves were prepared for light and electron microscopy. In a separate group, Fluro-Gold was injected subcutaneously at the ankle prior to perfusion, and counts and sizes of retrogradely labeled and unlabeled dorsal root ganglion neurons were obtained. There was no difference in the time course of gait recovery in antibody-treated and vehicle-treated animals. The number of myelinated and nonmyelinated axons was the same in the muMab 911-treated crushed nerves and intact nerves, consistent with observed complete recovery. Treatment with muMab 911 did however result in a small decrease in average cell body size on both the intact and injured sides. These results indicate that muMab 911 did not impair functional recovery or nerve regeneration after nerve injury in adult rats.

[The role of transplanted astrocytes for the regeneration of CNS axons].

Long tract axons in the mammalian CNS do not normally regenerate for appreciable distance after they transected. But we reported transplantation of Schwann cells(SCs) or olfactory ensheathing cells induced regeneration of transected rat dorsal column (DC) axons and improved the conduction. Scar formation(gliosis), for which astrocytes(ACs) play an important role, may be one of strong and physical barriers for the regeneration of CNS axon. Oligodendrocyte and myelin associated protein or products also inhibit the regeneration of the axons, as chemical barriers. To investigate how effective the promotion or the reduction of scar or myelin formation may be for axonal regeneration, we transplanted AC into transected DCs, or radiated(X-ray) the DCs, and compared to normal DCs or regenerated DCs following by SC transplantation. DCs of adult rats were transected at Th 11 and transplanted with SCs(6 x 10(4)) of adult rats or ACs(6 x 10(4)) of neonatal rats. Five to six weeks later, the spinal cords were removed and pinned in a recording chamber, and compound action potentials (CAPs) along the DC through the transected lesion were recorded, to investigate conduction properties(conduction velocity and response after high frequency stimulations). Following transplantation of SCs or ACs, histological examination revealed regenerated axons with SC-like patterns of remyelination in transected DCs. X-ray irradiation did not enhance the regeneration of DC axons. SC transplantation improved the conduction properties of transected DCs and increased the number of regenerated axons, compared to transected DCs without cell transplantation. AC transplantation resulted in improvement of the conduction properties, but the number of regenerated axons was similar to that of transected DCs without the transplantation. X-ray irradiation (40 Gy) three days before DC transection and AC transplantation prevented the electrophysiological continuity of axons through the transected lesion. This evidence revealed that AC transplantation secondarily enhanced the regeneration of axons, probably endogeneous SCs of dorsal roots migrated into the transected lesion and enhanced the axonal regeneration.

Transplantation of cryopreserved adult human Schwann cells enhances axonal conduction in demyelinated spinal cord.

Schwann cells derived from human sural nerve may provide a valuable source of tissue for a cell-based therapy in multiple sclerosis. However, it is essential to show that transplanted human Schwann cells can remyelinate axons in adult CNS and improve axonal conduction. Sections of sural nerve were removed from amputated legs of patients with vascular disease or diabetes, and Schwann cells were isolated and cryopreserved. Suspensions of reconstituted cells were transplanted into the X-irradiation/ethidium bromide lesioned dorsal columns of immunosuppressed Wistar rat. After 3-5 weeks of extensive remyelination, a typical Schwann cell pattern was observed in the lesion zone. Many cells in the lesion were immunopositive for an anti-human nuclei monoclonal antibody. The dorsal columns were removed and maintained in an in vitro recording chamber; the conduction properties were studied using field potential and intra-axonal recording techniques. The transplanted dorsal columns displayed improved conduction velocity and frequency-response properties, and action potentials conducted over a greater distance into the lesion, suggesting that conduction block was overcome. These data support the conclusion that transplantation of human Schwann cells results in functional remyelination of a dorsal column lesion.

Indications for therapeutic plasma exchange at a university hospital and a regional blood center.

The AABB guidelines for therapeutic plasma exchange (TPE) are divided into four categories: I. TPE is "standard and acceptable therapy," II. "generally accepted," III. "insufficient evidence to evaluate efficacy," and IV. "data suggest no therapeutic efficacy." Since little is known about the implementation of these guidelines, and since the indications for TPE may vary, depending upon an institution's patient mix, this study reviewed the indications and their categories for two co-located institutions. A retrospective review of the indications for all patients undergoing TPE from January 1, 1994 to December 31,1997 at Emory University Hospital (EUH), a tertiary-care teaching hospital, and the American Red Cross (ARC), a regional blood center, using AABB criteria (ASFA criteria used when not rated [NR] by AABB) was conducted. Categories I/II represented 75% and 88% of cases (EUH and ARC, respectively), while Categories III/IV/NR (NR as used below is "not rated" by both AABB and ASFA criteria; n is number of patients) were 25% and 12% of indications, respectively (P =0.002). Cases at EUH (n=101) were I, 62%; II, 13%; III, 3%; IV, 13%; and NR, 9%. Cases at ARC (n=359) were I, 77%; II, 11%; III, 9%; IV, 0%; and NR, 3% (P<0.001). No Category IV patients underwent TPE at ARC (13% at EUH). Thrombotic thrombocytopenic purpura (TTP) was the most common indication for TPE at both centers. The majority of the procedures were "appropriate" (Categories III/); several disorders ( approximately 10%) for which TPE was utilized at both centers were NR by both AABB and ASFA guidelines. Indications for TPE may differ, depending on the type of requesting institution. Physicians requesting TPE for patients with disorders in Categories III/IV/NR should be more strongly encouraged to enter their patients into controlled trials to best evaluate the efficacy of TPE in inadequately-studied clinical situations. This might best be accomplished at university hospitals, where requests for Category III/IV/NR may be higher. A need exists for periodic updating of the AABB guidelines to include those diseases for which new information is available with regard to the potential therapeutic role of TPE.

[Characteristic improvement of the function following Schwann cell transplantation for demyelinated spinal cord].

Transplantation of Schwann cells (SCs) induced remyelination of demyelinated rat dorsal column (DC) axons and improved conduction. To investigate the difference between oligodendrocyte (OL) and SC myelination in conductive functions of axons, we compared normal DCs, demyelinated DCs, demyelinated DCs remyelinated by SC transplantation, and normal dorsal roots. All of the axons was originated from dorsal root ganglion neurons. Dorsal roots of adult rats were demyelinated at T11 by X-ray irradiation and ethidium bromide, and transplanted with SCs (3 x 10(4)) of adult rats. Three weeks later, the spinal cord was removed and pinned in a recording chamber and compound action potentials (CAPs) were recorded, to investigate conduction properties (conduction velocity and response after high frequency stimulation). Normal DCs or dorsal roots were recorded in same manner. Following transplantation of SCs, histological examination revealed SC-like patterns of remyelination in demyelinated DCs. SC transplantation improved significantly conduction properties compared to demyelinated axons, but less than normal DC. Moreover, remyelinated axons by SC transplantation showed as low amplitude of CAP as dorsal roots, but lower conduction velocity than dorsal roots. Though anatomical difference and/or time after transplantation influenced the conduction, these result suggested that SC myelination resulted in lower amplitude of CAP than OL, and SC remyelination might be insufficient for conduction velocity.

Xenotransplantation of transgenic pig olfactory ensheathing cells promotes axonal regeneration in rat spinal cord.

Here we describe transplantation of olfactory ensheathing cells (OECs) or Schwann cells derived from transgenic pigs expressing the human complement inhibitory protein, CD59 (hCD59), into transected dorsal column lesions of the spinal cord of the immunosuppressed rat to induce axonal regeneration. Non-transplanted lesion-controlled rats exhibited no impulse conduction across the transection site, whereas in animals receiving transgenic pig OECs or Schwann cells impulse conduction was restored across and beyond the lesion site for more than a centimeter. Cell labeling indicated that the donor cells migrated into the denervated host tract. Conduction velocity measurements showed that the regenerated axons conducted impulses faster than normal axons. By morphological analysis, the axons seemed thickly myelinated with a peripheral pattern of myelin expected from the donor cell type. These results indicate that xenotranplantation of myelin-forming cells from pigs genetically altered to reduce the hyperacute response in humans are able to induce elongative axonal regeneration and remyelination and restore impulse conduction across the transected spinal cord.

[Comparison of myelin-forming cells as candidates for therapeutic transplantation in demyelinated CNS axons].

Demyelination of axons resulted in distinct reduction of conduction velocity or block of conduction. Remyelination by transplantation of myelin-forming cells may provide a therapeutic approach for demyelinated diseases. However, which cell type will be the most appropriate candidate for such a cell therapy is not established. To investigate how effective grafted neonatal brain cell (BC) (including oligodendrocyte and astrocyte) isolated from neonatal fronto-temporal lobes, adult olfactory ensheathing cell (OEC) or adult Schwann cell (SC) may be for demyelinated CNS axons in vivo, dorsal columns(DCs) of adult rat spinal cord were demyelinated at Th 11 by X-ray irradiation (day 0) and the injection of ethidium bromide (day 3), and transplanted 5 x 10(4) of BCs, 3 x 10(4) of OECs, or 3 x 10(4) of SCs into the lesion (day 6). Day 28-31, spinal cord were removed and transferred an in vitro recording chamber to record field potentials using glass micropipettes, to investigate conduction properties at 36 degrees. Normal DCs were recorded in same manner. Histological examination revealed that OECs and SCs resulted in substantial SC-like patterns of remyelination to equal degree, BC transplantation resulted in less myelination. The conduction velocities were significantly improved to 4.2 +/- 2.4 m/s(BC, n = 5), 8.5 +/- 3.3 m/s(OEC, n = 6) and 7.7 +/- 1.5 m/s(SC, n = 5), compared to demyelinated axons(1.2 +/- 0.4 m/s, n = 7). A 600 Hz 0.5 sec stimulus train led to an amplitude decrement of 7.1 +/- 7.5% (n = 7) in demyelinated axons. Following transplantation, the amplitude decreased in 31.3 +/- 18.7% (BC, n = 5), 49.9 +/- 19.9% (OEC, n = 6) and 66.2 +/- 11.9% (SC, n = 5). Transplanted OECs and SCs enhanced the remyelination of demyelinated CNS axons, and improved conduction properties were similar, and more effective than that induced from isolated CNS tissue which included oligodendrocyte.

Transplantation of olfactory ensheathing cells or Schwann cells restores rapid and secure conduction across the transected spinal cord.

Olfactory ensheathing cells (OECs) or Schwann cells were transplanted into the transected dorsal columns of the rat spinal cord to induce axonal regeneration. Electrophysiological recordings were obtained in an isolated spinal cord preparation. Without transplantation of cells, no impulse conduction was observed across the transection site; but following cell transplantation, impulse conduction was observed for over a centimeter beyond the lesion. Cell labelling indicated that the regenerated axons were derived from the appropriate neuronal source, and that donor cells migrated into the denervated host tract. As reported in previous studies, the number of regenerated axons was limited. Conduction velocity measurements and morphology indicated that the regenerated axons were myelinated, but conducted faster and had larger axon areas than normal axons. These results indicate that the regenerated spinal cord axons induced by cell transplantation provide a quantitatively limited but rapidly conducting new pathway across the transection site.

Effects of UVB radiation on cytokine generation, cell adhesion molecules, and cell activation markers in T-lymphocytes and peripheral blood HPCs.

BACKGROUND: Immunomodulatory effects of UV light have increasingly become a focus in transfusion medicine, BMT and transplantation immunology. In the transplant setting, the use of UVB radiation may reduce or abolish T-cell activation without compromising either bone marrow (BM) engraftment or graft-versus-leukemia effect. In this study, BM and apheresis-derived peripheral blood HPCs were used to investigate the effects of UVB on colony-forming ability, CD34+ cell viability, and growth potential, as well as on the secretion of MNC cytokines and the expression of cell surface markers and adhesion molecules. STUDY DESIGN AND METHODS: After UVB radiation, enriched populations of T cells and antigen-presenting cells (APCs) were treated with PHA, and the MNC response was measured, as was colony-forming ability. CD34+ cells were quantified and their growth potential was determined in culture. Next, T-cell activation status, cell adhesion molecule and cell surface activation marker expression, and cytokine profiles were evaluated, and cytokine mRNA was quantitated. Parallel studies were done in unirradiated control cell populations. RESULTS: Low-dose (10 mJ/cm(2)) UVB mitigates MNC proliferative responses by 94 percent while maintaining 60 and 80 percent of colony-forming ability in peripheral blood HPC and BM preparations, respectively, and >50 percent of colony-forming ability in CD34+ cell-enriched samples. Low-dose UVB radiation also significantly reduces T-cell production of TNFalpha, TNFalpha mRNA, TNFbeta, IL-2, and IL-6 and downregulates T-cell expression of CD28, CD25, CD69, and intercellular adhesion molecule 1. CONCLUSION: These findings have shown that a "window" of low-dose UVB radiation (10 mJ/cm(2)) exists, at which BM- and peripheral blood-derived MNC proliferation is inactivated, while the HPCs are relatively spared. UVB light selectively affects T cells, while APCs are resistant to low doses of UVB. UVB radiation also alters the expression of some cell surface markers and cytokines that are important in T-cell activation pathways. Reduction of T-cell activation without cytocidal effect may allow UVB radiation to become an immunomodulating agent in BM or HPC transplantation.

Utilization of fine-needle aspiration in the diagnosis of metastatic tumors to the kidney.

Renal masses secondary to metastases are not common. Few comprehensive reviews exist, which consist primarily of autopsy and radiologic reports. The purpose of this study was to review the types and incidences of various neoplasms which metastasize to the kidney and to determine the usefulness of fine-needle aspiration (FNA) in diagnosing them. Two hundred and sixty-one radiologically guided FNAs of renal lesions over a 9-yr period were reviewed. The diagnoses of the 261 renal FNAs were as follows: 136 (52%) were malignant, 111 (43%) were benign, and 14 (5%) were unsatisfactory. Of the 136 positive FNAs, 28 (21%) revealed metastatic tumors. The overall incidence of renal FNAs displaying metastatic tumors was 11%. Among the 28 patients with metastases to the kidney, 23 patients were men and 5 were women, with the mean age being 58 yr. Twenty-five patients (89%) had prior history of a primary malignancy, including lung carcinoma (11 cases, 39%), lymphoma (8 cases, 29%), hepatocellular carcinoma (3 cases, 11%), and one case each of breast, pancreatic, and cervical cancer. In the remaining 3 patients (11%), with metastatic adenocarcinoma (2 cases) and squamous-cell carcinoma (1 case), the primary tumor site remained unknown despite an extensive clinical workup. Overall survival after FNA was poor, with a mean of 9.8 mo. FNA is useful in the diagnosis of masses in the kidney secondary to metastatic disease. This information is of clinical importance, principally in the exclusion of a primary malignancy, but also to avoid unnecessary surgery and to plan for subsequent patient care.

Transplanted olfactory ensheathing cells remyelinate and enhance axonal conduction in the demyelinated dorsal columns of the rat spinal cord.

Olfactory ensheathing cells (OECs), which have properties of both astrocytes and Schwann cells, can remyelinate axons with a Schwann cell-like pattern of myelin. In this study the pattern and extent of remyelination and the electrophysiological properties of dorsal column axons were characterized after transplantation of OECs into a demyelinated rat spinal cord lesion. Dorsal columns of adult rat spinal cords were demyelinated by x-ray irradiation and focal injections of ethidium bromide. Cell suspensions of acutely dissociated OECs from neonatal rats were injected into the lesion 6 d after x-ray irradiation. At 21-25 d after transplantation of OECs, the spinal cords were maintained in an in vitro recording chamber to study the conduction properties of the axons. The remyelinated axons displayed improved conduction velocity and frequency-response properties, and action potentials were conducted a greater distance into the lesion, suggesting that conduction block was overcome. Quantitative histological analysis revealed remyelinated axons near and remote from the cell injection site, indicating extensive migration of OECs within the lesion. These data support the conclusion that transplantation of neonatal OECs results in quantitatively extensive and functional remyelination of demyelinated dorsal column axons.

Utilization of core wash material in the diagnosis of breast lesions by stereotactic needle biopsy.

BACKGROUND: Several reports have compared the results of fine-needle aspiration and stereotactic core needle biopsy in nonpalpable breast lesions. In this study the authors describe a simple method to retrieve cytologic material from a core breast biopsy sample that provides the diagnosis within 1 hour of the procedure. METHODS: Two hundred and eleven nonpalpable breast lesions were biopsied. Each core needle biopsy sample was placed in a mesh bag, and the bag and needle notch were washed in Cytolyt solution to obtain a monolayer using a commercial ThinPrep processor. The cytologic diagnoses were divided into four categories: benign, suspicious, malignant, and unsatisfactory, which then were compared with core needle biopsy results. RESULTS: Cytology reports of 211 lesions were as follows: 169 lesions (80%) were benign, 16 lesions (7.6%) were suspicious, 11 lesions (5.2%) were malignant, and 15 lesions (7.1%) were unsatisfactory. Core needle biopsy showed 165 of 169 samples (98%) to be benign and 4 to be malignant. Of the 16 suspicious smears, 10 were invasive carcinoma, 2 were in situ lesions, 3 were hyperplasias, and 1 was fibrosis. Of the 11 malignant smears, 10 were confirmed on core needle biopsy and 1 was read as atypia on the first core needle biopsy sample and malignant on a second, separate, follow-up core needle biopsy. Of the 15 unsatisfactory samples, 14 were found to be benign and 1 was found to be malignant on a separate, follow-up core needle biopsy. CONCLUSIONS: The core wash technique was 85% sensitive and 98% specific for malignancy. Only 7% of specimens were insufficient for diagnosis, and 93% of these were proven to be benign. This technique is useful for immediate (within 1 hour) diagnosis of breast lesions, alleviating patient anxiety and supplementing the diagnostic yield of the core biopsy.

Mechanisms of enhancement of neurite regeneration in vitro following a conditioning sciatic nerve lesion.

To examine the mechanisms responsible for the more rapid nerve regeneration observed after a previous (conditioning) nerve injury, adult rats were subjected to a midthigh sciatic nerve transection by using one of three protocols designed to facilitate or restrict nerve regeneration: 1) ligation, in which transected axons were prevented from regenerating; 2) cut, in which transected axons were permitted to extend into peripheral target tissue but were separated from the denervated peripheral nerve stump; and 3) crush, in which axons could regenerate normally through the denervated distal nerve tract. The affected dorsal root ganglia (DRG) were subsequently removed, dissociated, and cultured for up to 3 days, and the timing of neurite initiation, rate of outgrowth, and arborization pattern of previously injured neurons were compared with control DRG. Our results indicate that conditioning lesions have at least four distinct and differentially regulated effects on neuronal morphogenesis: 1) conditioning lesions promote earlier neurite initiation, 2) prior nerve injury decreases the ability of neurons to extend long neurites following a second axotomy, 3) exposure to the environment of a denervated peripheral nerve stimulates greater initial rates of neurite outgrowth, and 4) conditioning lesions reduces initial neuritic branching frequency, resulting in straighter neurites whose growth cones extend further distances from their cell bodies. The primary effect of all conditioning lesions on cultured DRG neurons appeared to be to advance the timing of morphogenesis, resulting in conditioning-lesioned neurons that exhibited characteristics consistent with control neurons that had been cultured for an additional day or more. A secondary effect of conditioning lesions on neurite outgrowth rates was dependent on the local environment of the axons prior to culturing.

Blocking Ca2+ mobilization with thapsigargin reduces neurite initiation in cultured adult rat DRG neurons.

Adult rat DRG neurons rapidly extend extensive neuritic arbors after a 1-2-day delay in culture and generate large depolarization-induced calcium signals during this time period that are derived primarily from intracellular calcium release. To assess whether intracellular calcium mobilization is required for neurite initiation, calcium stores were depleted by brief exposure to the irreversible endoplasmic reticulum calcium ATPase inhibitor thapsigargin; cultures were then maintained for 3 days, immunostained for neurofilament and scored for percentage of neurons with neurites at least twice as long as the cell body. Brief thapsigargin treatment (20 min) during the first 24 h in culture resulted in a substantial decrease in neurite initiation frequency without affecting neuronal or nonneuronal cell survival, suggesting that intracellular calcium mobilization is necessary for triggering neurite initiation in these neurons.

Intracellular calcium mobilization and neurite outgrowth in mammalian neurons.

Cultured adult rat dorsal root ganglion (DRG) neurons were used to study depolarization-induced Ca2+ mobilization and the effects of intracellular Ca2+ depletion on neurite outgrowth. Cytoplasmic and nuclear Ca2+ signals were visualized in dissociated DRG neurons using confocal scanning laser microscopy and the Ca2+ indicator dye fluo-3. The depolarization-induced Ca2+ signals were highest in neurons during the first few days in culture, prior to neurite extension; during this time nuclear signals exceeded those of the cytoplasm severalfold. After several days in culture, neurons began to arborize, depolarization-induced Ca2+ signals became attenuated, and nuclear signals no longer exceeded those of the cytoplasm. Elevated Ca2+ signals were dependent upon both Ca2+ influx and intact intracellular Ca2+ stores, indicating that the signals are generated by calcium-induced calcium release (CICR). Thapsigargin, an endoplasmic reticulum Ca2+ ATPase inhibitor, depleted intracellular Ca2+ stores and blocked the induction of the large nuclear Ca2+ signals. Treating DRG neurons briefly with thapsigargin (200 nM for 20 min) shortly after plating reduced subsequent neuritogenesis, implying that intact Ca2+ stores are necessary for initiating neurite outgrowth. Immunostaining of DRG neurons with antibodies to Ca2+/calmodulin-dependent kinase II (CaM kinase II) demonstrated that this enzyme is present in the nucleus at early times in culture. These observations are consistent with the idea that CICR triggered by Ca2+ entry subsequent to depolarization may elicit neurite outgrowth by activating nuclear enzymes appropriate for such outgrowth.