Intraoperative fluorescence imaging with aminolevulinic acid detects grossly occult breast cancer: a phase II randomized controlled trial.

BACKGROUND: Re-excision due to positive margins following breast-conserving surgery (BCS) negatively affects patient outcomes and healthcare costs. The inability to visualize margin involvement is a significant challenge in BCS. 5-Aminolevulinic acid hydrochloride (5-ALA HCl), a non-fluorescent oral prodrug, causes intracellular accumulation of fluorescent porphyrins in cancer cells. This single-center Phase II randomized controlled trial evaluated the safety, feasibility, and diagnostic accuracy of a prototype handheld fluorescence imaging device plus 5-ALA for intraoperative visualization of invasive breast carcinomas during BCS. METHODS: Fifty-four patients were enrolled and randomized to receive no 5-ALA or oral 5-ALA HCl (15 or 30 mg/kg). Forty-five patients (n = 15/group) were included in the analysis. Fluorescence imaging of the excised surgical specimen was performed, and biopsies were collected from within and outside the clinically demarcated tumor border of the gross specimen for blinded histopathology. RESULTS: In the absence of 5-ALA, tissue autofluorescence imaging lacked tumor-specific fluorescent contrast. Both 5-ALA doses caused bright red tumor fluorescence, with improved visualization of tumor contrasted against normal tissue autofluorescence. In the 15 mg/kg 5-ALA group, the positive predictive value (PPV) for detecting breast cancer inside and outside the grossly demarcated tumor border was 100.0% and 55.6%, respectively. In the 30 mg/kg 5-ALA group, the PPV was 100.0% and 50.0% inside and outside the demarcated tumor border, respectively. No adverse events were observed, and clinical feasibility of this imaging device-5-ALA combination approach was confirmed. CONCLUSIONS: This is the first known clinical report of visualization of 5-ALA-induced fluorescence in invasive breast carcinoma using a real-time handheld intraoperative fluorescence imaging device. TRIAL REGISTRATION: Clinicaltrials.gov identifier NCT01837225 . Registered 23 April 2013.

The Necrosis-Avid Small Molecule HQ4-DTPA as a Multimodal Imaging Agent for Monitoring Radiation Therapy-Induced Tumor Cell Death.

PURPOSE: Most effective antitumor therapies induce tumor cell death. Non-invasive, rapid and accurate quantitative imaging of cell death is essential for monitoring early response to antitumor therapies. To facilitate this, we previously developed a biocompatible necrosis-avid near-infrared fluorescence (NIRF) imaging probe, HQ4, which was radiolabeled with 111Indium-chloride (111In-Cl3) via the chelate diethylene triamine pentaacetic acid (DTPA), to enable clinical translation. The aim of the present study was to evaluate the application of HQ4-DTPA for monitoring tumor cell death induced by radiation therapy. Apart from its NIRF and radioactive properties, HQ4-DTPA was also tested as a photoacoustic imaging probe to evaluate its performance as a multimodal contrast agent for superficial and deep tissue imaging. MATERIALS AND METHODS: Radiation-induced tumor cell death was examined in a xenograft mouse model of human breast cancer (MCF-7). Tumors were irradiated with three fractions of 9 Gy each. HQ4-DTPA was injected intravenously after the last irradiation, NIRF and photoacoustic imaging of the tumors were performed at 12, 20, and 40 h after injection. Changes in probe accumulation in the tumors were measured in vivo, and ex vivo histological analysis of excised tumors was performed at experimental endpoints. In addition, biodistribution of radiolabeled [111In]DTPA-HQ4 was assessed using hybrid single-photon emission computed tomography-computed tomography (SPECT-CT) at the same time points. RESULTS: In vivo NIRF imaging demonstrated a significant difference in probe accumulation between control and irradiated tumors at all time points after injection. A similar trend was observed using in vivo photoacoustic imaging, which was validated by ex vivo tissue fluorescence and photoacoustic imaging. Serial quantitative radioactivity measurements of probe biodistribution further demonstrated increased probe accumulation in irradiated tumors. CONCLUSION: HQ4-DTPA has high specificity for dead cells in vivo, potentiating its use as a contrast agent for determining the relative level of tumor cell death following radiation therapy using NIRF, photoacoustic imaging and SPECT in vivo. Initial preclinical results are promising and indicate the need for further evaluation in larger cohorts. If successful, such studies may help develop a new multimodal method for non-invasive and dynamic deep tissue imaging of treatment-induced cell death to quantitatively assess therapeutic response in patients.

Emerging Applications for Optically Enabled Intravital Microscopic Imaging in Radiobiology.

Radiation therapy is an effective cancer treatment used in over 50% of cancer patients. Preclinical research in radiobiology plays a major role in influencing the translation of radiotherapy-based treatment strategies into clinical practice. Studies have demonstrated that various components of tumors and their microenvironments, including vasculature, immune and stem cells, and stromal cells, can influence the response of solid tumors to radiation. Optically enabled imaging techniques used in experimental animal models of cancer offer a unique and powerful way to quantitatively track spatiotemporal changes in these tumor components in vivo at macro-, meso-, and microscopic resolutions following radiotherapy. In this review, we discuss the role of both well-established and emerging intravital microscopy techniques for studying tumors and their microenvironment in vivo, in response to irradiation. The development and application of new animal models, small animal microirradiation technologies, and multimodal optically enabled intravital microscopy techniques are emphasized within the framework of preclinical radiobiology research. We also comment on the potential influence that these newer imaging techniques may have on the clinical translation of new preclinical radiobiology discoveries.

Point-of-care autofluorescence imaging for real-time sampling and treatment guidance of bioburden in chronic wounds: first-in-human results.

BACKGROUND: Traditionally, chronic wound infection is diagnosed by visual inspection under white light and microbiological sampling, which are subjective and suboptimal, respectively, thereby delaying diagnosis and treatment. To address this, we developed a novel handheld, fluorescence imaging device (PRODIGI) that enables non-contact, real-time, high-resolution visualization and differentiation of key pathogenic bacteria through their endogenous autofluorescence, as well as connective tissues in wounds. METHODS AND FINDINGS: This was a two-part Phase I, single center, non-randomized trial of chronic wound patients (male and female, ≥18 years; UHN REB #09-0015-A for part 1; UHN REB #12-5003 for part 2; clinicaltrials.gov Identifier: NCT01378728 for part 1 and NCT01651845 for part 2). Part 1 (28 patients; 54% diabetic foot ulcers, 46% non-diabetic wounds) established the feasibility of autofluorescence imaging to accurately guide wound sampling, validated against blinded, gold standard swab-based microbiology. Part 2 (12 patients; 83.3% diabetic foot ulcers, 16.7% non-diabetic wounds) established the feasibility of autofluorescence imaging to guide wound treatment and quantitatively assess treatment response. We showed that PRODIGI can be used to guide and improve microbiological sampling and debridement of wounds in situ, enabling diagnosis, treatment guidance and response assessment in patients with chronic wounds. PRODIGI is safe, easy to use and integrates into the clinical workflow. Clinically significant bacterial burden can be detected in seconds, quantitatively tracked over days-to-months and their biodistribution mapped within the wound bed, periphery, and other remote areas. CONCLUSIONS: PRODIGI represents a technological advancement in wound sampling and treatment guidance for clinical wound care at the point-of-care. TRIAL REGISTRATION: ClinicalTrials.gov NCT01651845; ClinicalTrials.gov NCT01378728.

Autofluorescence imaging device for real-time detection and tracking of pathogenic bacteria in a mouse skin wound model: preclinical feasibility studies.

Bacterial infection significantly impedes wound healing. Clinical diagnosis of wound infections is subjective and suboptimal, in part because bacteria are invisible to the naked eye during clinical examination. Moreover, bacterial infection can be present in asymptomatic patients, leading to missed opportunities for diagnosis and treatment. We developed a prototype handheld autofluorescence (AF) imaging device (Portable Real-time Optical Detection, Identification and Guidance for Intervention - PRODIGI) to noninvasively visualize and measure bacterial load in wounds in real time. We conducted preclinical pilot studies in an established nude mouse skin wound model inoculated with bioluminescent Staphylococcus aureus bacteria. We tested the feasibility of longitudinal AF imaging for in vivo visualization of bacterial load in skin wounds, validated by bioluminescence imaging. We showed that bacteria (S. aureus), occult to standard examination, can be visualized in wounds using PRODIGI. We also detected quantitative changes in wound bacterial load over time based on the antibiotic treatment and the correlation of bacterial AF intensity with bacterial load. AF imaging of wounds offers a safe, noninvasive method for visualizing the presence, location, and extent of bacteria as well as measuring relative changes in bacterial load in wounds in real time.

Potential of magnetic resonance-guided focused ultrasound for intracranial hemorrhage: an in vivo feasibility study.

BACKGROUND: Because of the paucity of effective treatments for intracranial hemorrhage (ICH), the mortality rate remains at 40%-60%. A novel application of magnetic resonance-guided focused ultrasound (MRgFUS) for ICH may offer an alternative noninvasive treatment through the precise delivery of FUS under real-time MR imaging (MRI) guidance. The purpose of the present study was to optimize the parameters for rapid, effective, and safe trans-skull large clot liquefaction using in vivo porcine and ex vivo human skull models to provide a clinically relevant proof of concept. METHODS: The transcranial effectiveness of MRgFUS was tested ex vivo by introducing a porcine blood clot into a human skull, without introducing tissue plasminogen activator (tPA). We used an experimental human head device to deliver pulsed FUS sonications at an acoustic power of 600-900 W for 5-10 seconds. A 3-mL clot was also introduced in a porcine brain and sonicated in vivo with one 5-second pulse of 700 W through a bone window or with 3000 W when treated through an ex vivo human skull. Treatment targeting was guided by MRI, and the tissue temperature was monitored online. Liquefied volumes were measured as hyperintense regions on T2-weighted MR images. RESULTS: In both in vivo porcine blood clot through a craniectomy model and the porcine clot in an ex vivo human skull model targeted clot liquefaction was achieved, with only marginal increase in temperature in the surrounding tissue. CONCLUSIONS: Our results demonstrate the feasibility of fast, efficient, and safe thrombolysis in an in vivo porcine model of ICH and in 2 ex vivo models using a human skull, without introducing tPA. Future studies will further optimize parameters and assess the nature of sonication-mediated versus natural clot lysis, the risk of rebleeding, the potential effect on the adjacent parenchyma, and the chemical and toxicity profiles of resulting lysate particles.

Potential of magnetic resonance-guided focused ultrasound for intracranial hemorrhage: an in vitro feasibility study.

BACKGROUND: Intracranial hemorrhage has a mortality rate of up to 40-60% due to the lack of effective treatment. Magnetic resonance-guided focused ultrasound may offer a breakthrough noninvasive technology, by allowing accurate delivery of focused ultrasound, under the guidance of real-time magnetic resonance imaging. AIM: The purpose of the current study was to optimize the acoustic parameters of magnetic resonance-guided focused ultrasound for effective clot liquefaction, in order to evaluate the feasibility of magnetic resonance-guided focused ultrasound for thrombolysis. METHODS: Body (1·1 MHz) and brain (220 kHz) magnetic resonance-guided focused ultrasound systems (InSightec Ltd, Tirat Carmel, Israel) were used to treat tube-like (4 cc), round (10 cc), and bulk (300 cc) porcine blood clots in vitro, using burst sonications of one-second to five-seconds, a duty cycle of 5-50%, and peak acoustic powers between 600 and 1200 W. Liquefied volumes were measured as hyperintense regions on T2-weighted magnetic resonance images for body unit sonications (duration of one-second, duty cycle of 10%, and power of 500-1200 W). Liquefaction efficiency was calculated for brain unit sonications (duration of one-second, duty cycle of 10%, power of 600 W, and burst length between 0·1 ms and 100 ms). RESULTS: Liquified lesion volume increased as power was raised, without a thermal rise. For brain unit sonications, a power setting of 600 W and ultrashort sonications (burst length between 0·1 and 1·0 ms) resulted in liquefaction efficacy above 50%, while longer burst duration yielded lower efficacy. CONCLUSIONS: These results demonstrate the feasibility of obtaining reproducible, rapid, efficient, and accurate blood clot lysis using the magnetic resonance-guided focused ultrasound system. Further in vivo studies are needed to validate the feasibility of magnetic resonance-guided focused ultrasound as a treatment modality for intracranial hemorrhage.

Endogenous radial glial cells support regenerating axons after spinal cord transection.

During the development of central nervous system, radial glial cells support target-specific neuronal migration. We recently reported that after implantation of chitosan channels with complete spinal cord transection, the tissue bridging the spinal cord stumps contained axons and radial glial cells. The purpose of this study was to clarify the role of the radial glial cells in the tissue bridges. Chitosan channels were implanted in rats with thoracic spinal cord transection. After 14 weeks, all animals had tissue bridges in the channels that contained many radial glial cells in longitudinal arrangement, some of which were in contact with axons in the bridges. We suggest that radial glial cells can guide regenerating axons across the bridge in the channel after spinal cord transection.

Stem/precursor cell-based CNS therapy: the importance of circumventing immune suppression by transplanting autologous cells.

Stem/precursor cell (SPC) therapy for neurodegeneration and neurotrauma has enormous therapeutic potential, but despite ongoing research efforts the success of clinical trials remains limited. Therapies that utilize immune suppression in combination with SPC transplantation have thus far failed to consider the beneficial role of the immune system in central nervous system (CNS) recovery. Systemic immune suppression may prevent neural repair, and in some cases exacerbate the underlying disorder. Until about a decade ago, immunosuppression for CNS disorders was viewed as a therapeutic target, based on the perception that all immune activity in the CNS was destructive. However, recent studies show that the infiltration of blood-borne immune cells into the CNS following neurotrauma and during chronic neurodegeneration promote CNS protection and regeneration. In the context of SPC therapies, although immune suppression prevents rejection of non-autologous cell grafts, it also prevents the restorative immune response by eliminating the immune mediated guidance cues that are required for SPCs to migrate to the location they are needed, and preventing SPC-mediated immunomodulation. This article argues in favor of transplanting autologous SPCs, particularly bone marrow derived cells. The therapeutic use of autologous SPCs for neural repair circumvents the need for concomitant immune suppression, exploits the immunomodulatory capacity of these cells, and maintains the immune niche that supports neural repair and is required to guide these cells to their appropriate locations. Overall, such an approach accommodates the requirements for translational therapeutics, and provides a standardized platform for reconciling the inherent controversies in the science.

Region-specific differentiation potential of adult rat spinal cord neural stem/precursors and their plasticity in response to in vitro manipulation.

This study characterized the differentiation of neural stem/precursor cells (NSPCs) isolated from different levels of the spinal cord (cervical vs lumbar cord) and different regions along the neuraxis (brain vs cervical spinal cord) of adult male Wistar enhanced green fluorescent protein rats. The differentiation of cervical spinal cord NSPCs was further examined after variation of time in culture, addition of growth factors, and changes in cell matrix and serum concentration. Brain NSPCs did not differ from cervical cord NSPCs in the percentages of neurons, astrocytes, or oligodendrocytes but produced 26.9% less radial glia. Lumbar cord NSPCs produced 30.8% fewer radial glia and 6.9% more neurons compared with cervical cord NSPCs. Spinal cord NSPC differentiation was amenable to manipulation by growth factors and changes in in vitro conditions. This is the first study to directly compare the effect of growth factors, culturing time, serum concentration, and cell matrix on rat spinal cord NSPCs isolated, propagated, and differentiated under identical conditions.

Adult spinal cord stem/progenitor cells transplanted as neurospheres preferentially differentiate into oligodendrocytes in the adult rat spinal cord.

Neural stem/progenitor cells (NSPCs) capable of generating new neurons and glia reside in the adult mammalian spinal cord. Transplantation of NSPCs has therapeutic potential for spinal cord injury, although there is limited information on the ability of these cells to survive and differentiate in vivo. Neurospheres cultured from the periventricular region of the adult spinal cord contain NSPCs that are self-renewing and multipotent. We examined the survival, proliferation, migration, and differentiation of adult spinal cord NSPCs generated from green fluorescent protein (GFP) transgenic rats and transplanted into the intact spinal cord. The grafted GFP-expressing cells survived for at least 6 weeks in vivo and migrated from the injection site along the rostro-caudal axis of the spinal cord. Transplanted cells transiently proliferated following transplantation and approximately 17% of the GFP-positive cells were apoptotic at 1 day. Also, better survival was seen with NSPCs transplanted as neurospheres in comparison to NSPCs transplanted as dissociated cells. By 1 week posttransplantation, grafted cells primarily expressed an oligodendrocytic phenotype and only 2% differentiated into astrocytes. Approximately 75% versus 38% of the grafted cells differentiated into oligodendrocytes after transplantation into spinal white versus gray matter, respectively. This is the first report to examine the time course of cell survival, proliferation, apoptosis, and phenotypic differentiation of transplanted NSPSs in the spinal cord. This is also the first report to examine the differences between transplanted NSPCs grafted as neurospheres or dissociated cells, and to compare the differentiation potential after transplantation into spinal cord white versus gray matter.

Glial precursor cell transplantation therapy for neurotrauma and multiple sclerosis.

Traumatic injury to the brain or spinal cord and multiple sclerosis (MS) share a common pathophysiology with regard to axonal demyelination. Despite advances in central nervous system (CNS) repair in experimental animal models, adequate functional recovery has yet to be achieved in patients in response to any of the current strategies. Functional recovery is dependent, in large part, upon remyelination of spared or regenerating axons. The mammalian CNS maintains an endogenous reservoir of glial precursor cells (GPCs), capable of generating new oligodendrocytes and astrocytes. These GPCs are upregulated following traumatic or demyelinating lesions, followed by their differentiation into oligodendrocytes. However, this innate response does not adequately promote remyelination. As a result, researchers have been focusing their efforts on harvesting, culturing, characterizing, and transplanting GPCs into injured regions of the adult mammalian CNS in a variety of animal models of CNS trauma or demyelinating disease. The technical and logistic considerations for transplanting GPCs are extensive and crucial for optimizing and maintaining cell survival before and after transplantation, promoting myelination, and tracking the fate of transplanted cells. This is especially true in trials of GPC transplantation in combination with other strategies such as neutralization of inhibitors to axonal regeneration or remyelination. Overall, such studies improve our understanding and approach to developing clinically relevant therapies for axonal remyelination following traumatic brain injury (TBI) or spinal cord injury (SCI) and demyelinating diseases such as MS.

Extramedullary chitosan channels promote survival of transplanted neural stem and progenitor cells and create a tissue bridge after complete spinal cord transection.

Transplantation of neural stem and progenitor cells (NSPCs) is a promising strategy for repair after spinal cord injury. However, the epicenter of the severely damaged spinal cord is a hostile environment that results in poor survival of the transplanted NSPCs. We examined implantation of extramedullary chitosan channels seeded with NSPCs derived from transgenic green fluorescent protein (GFP) rats after spinal cord transection (SCT). At 14 weeks, we assessed the survival, maturation, and functional results using NSPCs harvested from the brain (brain group) or spinal cord (SC group) and seeded into chitosan channels implanted between the cord stumps after complete SCT. Control SCT animals had empty chitosan channels or no channels implanted. Channels seeded with brain or spinal cord-derived NSPCs showed a tissue bridge, although the bridges were thicker in the brain group. Both cell types showed long-term survival, but the number of surviving cells in the brain group was approximately five times as great as in the SC group. In both the brain and SC groups at 14 weeks after transplantation, many host axons were present in the center of the bridge in association with the transplanted cells. At 14 weeks astrocytic and oligodendrocytic differentiation in the channels was 24.8% and 17.3%, respectively, in the brain group, and 31.8% and 9.7%, respectively, in the SC group. The channels caused minimal tissue reaction in the adjacent spinal cord. There was no improvement in locomotor function. Thus, implantation of chitosan channels seeded with NSPCs after SCT created a tissue bridge containing many surviving transplanted cells and host axons, although there was no functional improvement.

Fate of transplanted adult neural stem/progenitor cells and bone marrow-derived mesenchymal stromal cells in the injured adult rat spinal cord and impact on functional recovery.

BACKGROUND: Neural stem/progenitor cells derived from the ependymal region of the spinal cord may have the ability to regenerate the injured mammalian spinal cord as they do in some lower vertebrates. It has also been suggested that BMSCs provide an environment conducive to regeneration in the injured cord. METHODS: In the current study, both spinal cord-derived NSPCs and BMSCs were cultured from adult male rats expressing eGFP. Neurospheres or dissociated BMSCs were transplanted 9 days after clip compression injury (35-g force). Cell survival and fate, and functional recovery were examined after 14 weeks. RESULTS: BMSCs showed no neural differentiation but had much better survival than NSPCs. Transplanted NSPCs differentiated mainly into astrocytes (14.7%) and oligodendrocytes (34.7%), but no neurons. No functional improvement was seen in either transplant group. However, in the NSPC group there was a significant inverse correlation between the functional scores and the number of transplanted astrocytes. A separate experiment to test the effect of cyclosporine on survival and fate of transplanted NSPCs showed that high-dose (20 mg/kg per day) cyclosporine improved cell survival, but had no effect on cell fate. CONCLUSIONS: Further work is required before these transplantation strategies can be recommended for patients. These results are promising in that we have found potentially beneficial mechanisms of action of the transplanted cells including differentiation of many NSPCs into oligodendrocytes with the possibility of promoting remyelination, and potential axonal guidance through guiding strands of matrix generated by the BMSCs.

Transplantation of adult rat spinal cord stem/progenitor cells for spinal cord injury.

Stem/progenitor cells derived from the ependymal region of the spinal cord have the ability to self-renew and are multipotential for neurons and glia. These cells may have the ability to regenerate the injured mammalian spinal cord as they do in some lower vertebrates. However, the optimal conditions for transplantation and the fate of transplanted cells are not fully known. In the current study, spinal cord stem/progenitor cells were cultured from adult male rats expressing enhanced green fluorescent protein (eGFP). Neurospheres were transplanted at the time of clip compression injury (35-g force) into the injury site, or 1 mm rostral and caudal to the injury site. Neurospheres were also transplanted into a subacute model (day 9 after injury) and a chronic model (day 28 after injury). Functional recovery was also studied in an acute injury model with weekly locomotor testing over a 16-week period. A significant increase in cell survival at 7 days was seen in rats receiving rostral and caudal injections as compared to injection directly into the site of injury. A significant increase in cell survival was also seen in rats receiving subacute transplants at 9 days after injury. Transplanted cells differentiated primarily into astrocytes (31.2%) and oligodendrocytes (50.3%), and a small number of neurons (1%). No improvement was seen in the Basso, Beattie and Bresnahan (BBB) locomotor rating scale after acute transplantation as compared with injury only, although surviving transplanted cells were identified that had migrated across the injury site from the rostral and caudal injection sites.

Oligodendrocytes and radial glia derived from adult rat spinal cord progenitors: morphological and immunocytochemical characterization.

Self-renewing, multipotent neural progenitor cells (NPCs) reside in the adult mammalian spinal cord ependymal region. The current study characterized, in vitro, the native differentiation potential of spinal cord NPCs isolated from adult enhanced green fluorescence protein rats. Neurospheres were differentiated, immunocytochemistry (ICC) was performed, and the positive cells were counted as a percentage of Hoescht+ nuclei in 10 random fields. Oligodendrocytes constituted most of the NPC progeny (58.0% of differentiated cells; 23.4% in undifferentiated spheres). ICC and electron microscopy (EM) showed intense myelin production by neurospheres and progeny. The number of differentiated astrocytes was 18.0%, but only 2.8% in undifferentiated spheres. The number of differentiated neurons was 7.4%, but only 0.85% in undifferentiated spheres. The number of differentiated radial glia (RG) was 73.0% and in undifferentiated spheres 80.9%. EM showed an in vitro phagocytic capability of NPCs. The number of undifferentiated NPCs was 32.8% under differentiation conditions and 78.9% in undifferentiated spheres. Compared with ependymal region spheres, the spheres derived from the peripheral white matter of the spinal cord produced glial-restricted precursors. These findings indicate that adult rat spinal cord ependymal NPCs differentiate preferentially into oligodendrocytes and RG, which may support axonal regeneration in future trials of transplant therapy for spinal cord injury.

Bone regeneration with active angiogenesis by basic fibroblast growth factor gene transfected mesenchymal stem cells seeded on porous beta-TCP ceramic scaffolds.

Large segmental bone defect repair remains a clinical and scientific challenge with increasing interest focused on combining gene transfer with tissue engineering techniques. Basic fibroblast growth factor (bFGF) is one of the most prominent osteogenic growth factors that has the potential to accelerate bone healing by promoting the proliferation and differentiation of mesenchymal stem cells (MSCs) and the regeneration of capillary vasculature. However, the short biological half-lives of growth factors may impose severe restraints on their clinical usefulness. Gene-based delivery systems provide a better way of achieving a sustained high concentration of growth factors locally in the defect and delivering a more biologically active product than that achieved by exogenous application of recombinant proteins. The objective of this experimental study was to investigate whether the bFGF gene modified MSCs could enhance the repair of large segmental bone defects. The pcDNA3-bFGF gene transfected MSCs were seeded on biodegradable porous beta tricalcium phosphate (beta-TCP) ceramics and allografted into the 15 mm critical-sized segmental bone defects in the radius of 18 New Zealand White rabbits. The pcDNA3 vector gene transfected MSCs were taken as the control. The follow-up times were 2, 4, 6, 8, 10 and 12 weeks. Scanning electron microscopic, roentgenographic, histologic and immunohistological studies were used to assess angiogenesis and bone regeneration. In vitro, the proliferation and differentiation of bFGF gene transfected MSCs were more active than that of the control groups. In vivo, significantly more new bone formation accompanied by abundant active capillary regeneration was observed in pores of the ceramics loaded with bFGF gene transfected MSCs, compared with control groups. Transfer of gene encoding bFGF to MSCs increases their osteogenic properties by enhancing capillary regeneration, thus providing a rich blood supply for new bone formation. This new bFGF gene enhanced tissue engineering strategy could be of potential benefit to accelerate bone healing, especially in defects caused by atrophic nonunion and avascular necrosis of the femoral head.

Analysis of green fluorescent protein expression in transgenic rats for tracking transplanted neural stem/progenitor cells.

Green fluorescent protein (GFP) expression was evaluated in tissues of different transgenic rodents--Sprague-Dawley (SD) rat strain [SD-Tg(GFP)Bal], W rat strain [Wistar-TgN(CAG-GFP)184ys], and M mouse strain [Tg(GFPU)5Nagy/J]--by direct fluorescence of native GFP expression and by immunohistochemistry. The constitutively expressing GFP transgenic strains showed tissue-specific differences in GFP expression, and GFP immunohistochemistry amplified the fluorescent signal. The fluorescence of stem/progenitor cells cultured as neurospheres from the ependymal region of the adult spinal cord from the GFP SD and W rat strains was assessed in vitro. After transplantation of the cells into wild-type spinal cord, the ability to track the grafted cells was evaluated in vivo. Cultured stem/progenitor cells from the SD strain required GFP immunostaining to be visualized. Likewise, after transplantation of SD cells into the spinal cord, immunohistochemical amplification of the GFP signal was required for detection. In contrast, GFP expression of stem/progenitor cells generated from the W strain was readily detected by direct fluorescence both in vitro and in vivo without the need for immunohistochemical amplification. The cultured stem/progenitor cells transplanted into the spinal cord survived for at least 49 days after transplantation, and continued to express GFP, demonstrating stable expression of the GFP transgene in vivo.

Calcium entry through L-type calcium channels is essential for neurite regeneration in cultured sympathetic neurons.

Previous work showed that a post-neuritotomy rise in [Ca2+]i is required for regeneration. We tested the following hypotheses in cultured sympathetic neurons: (1) blocking L-type channels at the time of injury inhibits regeneration; (2) enhancing Ca2+ entry through L-type Ca2+ channels enhances regeneration; (3) L-type Ca2+ channel distribution is predominantly on the soma and proximal neurites of uninjured and injured neurons. To visualize L-type Ca2+ channels and block Ca2+ influx, the fluorescent dihydropyridine antagonist, DM-BODIPY, was used. Our results show that regeneration is markedly inhibited by the antagonist when administered 20 min. prior to injury, in the presence or absence of nerve growth factor (NGF) (p < 0.0001). Severe degeneration of proximal and distal neurites was seen 48 h after injury. Regeneration was minimally inhibited by the antagonist when administered 5 min after injury (p < 0.05), but not inhibited when administered 2 or 24 h after injury (p > 0.05). We found that L-type channels are distributed ubiquitously on the soma and neurites of uninjured and injured cells, and on regenerating neurites. The addition of the L-type channel agonist, BayK8644, (1 microM) 20 min prior to injury enhanced neurite length at 24 h post-injury (p = 0.002). Blocking L-type channels did not affect the viability of uninjured or injured cells. For the first time, it has been shown that Ca2+ entry through L-type Ca2+ channels is essential for post-neuritotomy sympathetic neurite regeneration, and that this effect shows a strict temporal dependency. We also demonstrated that regeneration can be enhanced by increasing Ca2+ influx through L-type channels.

Inosine reverses the inhibitory effects of the L-type Ca2+ channel antagonist, DM-BODIPY-dihydropyridine, on neuritogenesis in an in vitro rat superior cervical ganglia axotomy model.

It has recently been demonstrated that L-type calcium channel antagonism with the fluorescent dihydropyridine DM-BODIPY-dihydropyridine (DMBD) inhibits neurite regeneration in rat superior cervical ganglia (SCG). The neuritogenic effects of inosine have been described in various models and the mechanism is thought to be N-kinase dependent. Because of the final common pathway between calcium dependent and N-kinase dependent neurite regeneration it was hypothesized that inosine would increase regeneration in normally regenerating SCG and reverse the inhibitory effects of DMBD on regenerating SCG. An in vitro model of rat SCG injury, where mature neurites are transected and observed at 2 and 24 h, was used to assess the effects of inosine on DMBD treated neurons. Results demonstrate a significant inhibition of growth in DMBD treated cultures, significantly increased growth in the inosine + DMBD treated SCG over DMBD treated cells and significantly increased growth in the inosine alone treated group over control cells. There is also evidence that inosine + DMBD treatment promotes linear growth of neurites. The implications of the findings are discussed.