carbocyanines and Spinal-Cord-Injuries

The possibility to control the fate of the cells responsible for secondary mechanisms following spinal cord injury (SCI) is one of the most relevant challenges to reduce the post traumatic degeneration of the spinal cord. In particular, microglia/macrophages associated inflammation appears to be a self-propelling mechanism which leads to progressive neurodegeneration and development of persisting pain state. In this study we analyzed the interactions between poly(methyl methacrylate) nanoparticles (PMMA-NPs) and microglia/macrophages in vitro and in vivo, characterizing the features that influence their internalization and ability to deliver drugs. The uptake mechanisms of PMMA-NPs were in-depth investigated, together with their possible toxic effects on microglia/macrophages. In addition, the possibility to deliver a mimetic drug within microglia/macrophages was characterized in vitro and in vivo. Drug-loaded polymeric NPs resulted to be a promising tool for the selective administration of pharmacological compounds in activated microglia/macrophages and thus potentially able to counteract relevant secondary inflammatory events in SCI.

Topics: Animals; Behavior, Animal; Carbocyanines; Cell Survival; Cells, Cultured; Coloring Agents; Drug Carriers; Female; Hydrogels; Lipopolysaccharides; Macrophages; Mice; Mice, Inbred C57BL; Microglia; Nanoparticles; Polymethyl Methacrylate; Spinal Cord; Spinal Cord Injuries

Bone marrow stromal cells (BMSC) have been successfully employed for movement deficit recovery in spinal cord injury (SCI) rat models. One of the unsettled problems in cell transplantation is the relative high proportion of cell death, specifically after neural differentiation. According to our previous studies, p75 receptor, known as the death receptor, is only expressed in BMSC in a time window of 6-12 hours following neural induction. Moreover, we have recently reported a decreased level of apoptosis in p75-suppressed BMSC in vitro. Therefore, our objective in this research was to explore the functional effects of transplanting p75:siRNA expressing BMSC in SCI rats.. Laminectomy was performed at L1 vertebra level to expose spinal cord for contusion using weight-drop method. PBS-treated SCI rats (group one) were used as negative controls, in which cavitations were observed 10 weeks after SCI. pRNA-U6.1/Hygro- (group two, as a mock) and pRNA-U6.1/Hygro-p75 shRNA- (group three) transfected BMSC were labeled with a fluorescent dye, CM-DiI, and grafted into the lesion site 7 days after surgery. The Basso-Beattie-Bresnehan locomotor rating scale was performed weekly for 10 weeks.. There was a significant difference (P≤0.05) between all groups of treated rats regarding functional recovery. Specifically, the discrepancy among p75 siRNA and mock-transfected BMSC was statistically significant. P75 siRNA BMSC also revealed a higher level of in vivo survival compared to the mock BMSC.. Our data suggest that genetically modified BMSC that express p75:siRNA could be a more suitable source of cells for treatment of SCI.

Topics: Animals; Behavior, Animal; Carbocyanines; Cell Lineage; Cell Movement; Cell Survival; Disease Models, Animal; Female; Fluorescence; Mesenchymal Stem Cell Transplantation; Mesenchymal Stem Cells; Motor Activity; Rats; Rats, Sprague-Dawley; Receptor, Nerve Growth Factor; Recovery of Function; Spinal Cord Injuries; Staining and Labeling; Transfection

The authors describe a method for percutaneous transplantation of human umbilical cord blood (hUCB)-derived multipotent stem cells (MSCs) under fluoroscopic guidance. The investigators then tested whether percutaneous transplantation of hUCB-derived MSCs improved neurological functional recovery after acute spinal cord injury (SCI).. The authors induced SCI in 10 dogs by percutaneous balloon compression. The 10 injured dogs were assigned randomly to the following groups (2 dogs each): Group 1, evaluated 2 weeks after sham transplantation; Group 2, evaluated 2 weeks after transplantation; Group 3, evaluated 4 weeks after sham transplantation; Group 4, evaluated 4 weeks after transplantation; and Group 5, evaluated 4 weeks after multispot transplantations. The dogs with sham transplantation (Groups 1 and 3) received the same volume of saline, as a control. A spinal needle was advanced into the spinal canal, and the investigators confirmed that the end of the spinal needle was located in the ventral part of spinal cord parenchyma by using contrast medium under fluoroscopic guidance. The hUCB-derived MSCs were transplanted into the cranial end of the injured segment in 6 injured dogs at 7 days after SCI.. Two dogs in Group 2 showed no improvement until 2 weeks after transplantation. Three of 4 dogs (Groups 4 and 5) that received cellular transplants exhibited gradual improvement in hindlimb locomotion from 3 weeks after cell transplantation. The CM-DiI-labeled hUCB-derived MSCs were observed in the spinal cord lesions at 4 weeks posttransplantation and exerted a significant beneficial effect by reducing cyst and injury size. The transplanted cells were positive for NeuN, glial fibrillary acidic protein, and von Willebrand factor.. The percutaneous transplantation technique described here can be easily performed, and it differs from previous techniques by avoiding surgical exposure and allowing cells to be more precisely transplanted into the spinal cord. This technique has many potential applications in the treatment of human SCI by cell transplantation. The results also suggest that transplantation of hUCB-derived MSCs may have therapeutic effects that decrease cavitation for acute SCI.

Topics: Animals; Carbocyanines; Contrast Media; Cord Blood Stem Cell Transplantation; Disease Models, Animal; Dogs; Fluorescent Dyes; Fluoroscopy; Humans; Immunoenzyme Techniques; Multipotent Stem Cells; Random Allocation; Spinal Cord Injuries; Staining and Labeling

Although fluorescent dyes combined with flow cytometry have been used to confirm the viability of sperm in the past, methods to detect damage to spermatozoa following injury have been limited to use of dyes, which are often difficult to adequately compensate for in a single laser system.. In this article, we present what we believe is a better method to assess damage to sperm secondary to spinal cord injury in an in vivo model, for use with a standard Ar laser and flow cell. In this rat model of spinal cord injury leading to sperm damage, the spinal cords of the rats were injured, but the reproductive organs were not. To understand the origins of sperm injury, and to develop ways to overcome the loss of fertility, we used the viability dye SYBR-14 along with 7-amino actinomycin D to detect apoptosis. Additionally, we used the dye JC-1 to measure the changes in mitochondrial transmembrane potential that accompany the damage.. We found that SYBR-14 plus 7-amino actinomycin D was a useful method for quantifying apoptosis, particularly when another dye, such as JC-1, was used simultaneously. By using these dyes in concert with motility studies, we were able to quantify the extent of damage to sperm and correlate it to the decrease in motility of sperm (r(2) = 0.99 for SYBR14 versus motility and r(2) = 0.98 for JC-1 versus motility by regression analysis).. With a method established to measure injury to sperm, we hope to determine which treatment regimens of ones we will test are effective in restoring sperm to a more fertile state, in the future.

Topics: Animals; Benzimidazoles; Carbocyanines; Cell Survival; Dactinomycin; Flow Cytometry; Fluorescent Dyes; Lasers; Male; Organic Chemicals; Rats; Rats, Sprague-Dawley; Sperm Motility; Spermatozoa; Spinal Cord Injuries

The spinal cord of the eel, Anguilla, recovers function rapidly after it has been completely transected. At transection, the excitability of central pattern generating circuits in the distal denervated segments increases to such a level that undulatory movements can occur spontaneously. When this elevated neuronal activity was reduced locally, just caudal to the transection, by chronic blockade of the NMDA receptor, the normally rapid behavioural recovery was retarded. The NMDA-treated fish overcame cordotomy more slowly than untreated animals, and axons did not extend as far into the denervated cord as in untreated counterparts, until later stages of recovery. These results suggest that central pattern generating activity may facilitate axonal growth in spinal cord regeneration.

Topics: 2-Amino-5-phosphonovalerate; Anguilla; Animals; Axons; Behavior, Animal; Carbocyanines; Drug Implants; Evoked Potentials; Locomotion; Nerve Regeneration; Receptors, N-Methyl-D-Aspartate; Recovery of Function; Spinal Cord Injuries; Swimming; Time Factors

After transection, the spinal cord of the eel Anguilla quickly regrows and reconnects, and function recovers. We describe here the changes in the central canal region that accompany this regeneration by using serial semithin plastic sections and immunohistochemistry. The progress of axonal regrowth was followed in material labeled with DiI. The canal of the uninjured cord is surrounded by four cell types: S-100-immunopositive ependymocytes, S-100- and glial fibrillary acidic protein (GFAP)-immunopositive tanycytes, vimentin-immunopositive dorsally located cells, and lateral and ventral liquor-contacting neurons, which label for either gamma-aminobutyric acid (GABA) or tyrosine hydroxylase (TH). After cord transection, a new central canal forms rapidly as small groups of cells at the leading edges of the transection create flat "plates" that serve as templates for subsequent formation of the lateral and dorsal walls. Profile counts and 5-bromo-2'-deoxyuridine immunohistochemistry indicate that these cells are dividing rapidly during the first 20 days of the repair process. The newly formed canal, which bridges the transection by day 10 but is not complete until about day 20, is greatly enlarged (

Topics: Anguilla; Animals; Carbocyanines; Cell Division; Ependyma; gamma-Aminobutyric Acid; Glial Fibrillary Acidic Protein; Growth Cones; Immunohistochemistry; Nerve Regeneration; Neuronal Plasticity; S100 Proteins; Spinal Cord; Spinal Cord Injuries; Stem Cells; Tyrosine 3-Monooxygenase; Vimentin

Human amniotic epithelial cells (HAEC) possess certain properties similar to that of neural and glial cells. In the present work, the potential of HAEC as stem cells for spinal cord injury repair was tested. HAEC obtained from human placenta were labeled with 1,1'-dioctadecyl-3,3,3',3'-tetramethyllindocarbocyanine perchlorate (Dil) in the culture medium. These labeled cells were transplanted into the transection cavities in the spinal cord of bonnet monkeys. Results were analyzed after 15 and 60 days of post-transplantation. HAEC cells survived in the monkey spinal cord for up to the maximum period of observation in the present study, i.e. 60 days. HAEC graft was penetrated by the host axons. There was no glial scar at the transection lesion site. Some of the host spinal neurons and axons were labeled with Dil (used to label HAEC) whereas in lesion control group, there was no such host-neuron labeling. This may be either due to the prevention of death in the axotomized neuron's ensuing lesion or due to the neurotrophic effect exhibited by the transplanted HAEC. Further studies would be required to verify these speculations. Therefore from this pilot study it appears that HAEC survive in the transplanted environment, support the growth of host axons through them, prevent the formation of glial scar at the cut ends and may prevent death in axotomized cells or attract the growth of new collateral sprouting. The abovementioned properties, i.e. serving as a suitable milieu for the host axons to grow, preventing glial scar at the lesion site and rescuing axotomized neurons from death were previously reported in the case of neural transplantation studies. Thus it is speculated that HAEC may be having certain properties equal to the beneficial effects of neural tissue in repairing spinal cord injury. Apart from this speculation, there are two more reasons for why HAEC transplantation studies are warranted to understand the long-term effects of such transplantations. First, there was no evidence of immunological rejection probably due to the non-antigenic nature of the HAEC. Second, unlike neural tissue, procurement of HAEC does not involve many legal or ethical problems.

Topics: Amnion; Animals; Carbocyanines; Cell Communication; Cell Differentiation; Cells, Cultured; Epithelial Cells; Female; Gliosis; Graft Survival; Growth Cones; Humans; Macaca radiata; Nerve Degeneration; Nerve Regeneration; Neuroglia; Neurons; Pregnancy; Spinal Cord Injuries; Stem Cell Transplantation; Transplantation, Heterologous; Treatment Outcome

Transected dorsal root axons of adult rats can be induced to regenerate through the normally non-permissive environment of the dorsal root entry zone (DREZ) into the spinal cord by implanting enteric glia (EG) into the DREZ. We have now examined whether the regenerating central axons make functional connections by studying the return of function of a behavioral response, the cutaneous trunci muscle (CTM) reflex. Implantation of EG into the spinal cord DREZ led to functional recovery of the CTM reflex in 82%, 72% and 70% of animals 1, 2 and 3 months, respectively, after injury. In contrast, the CTM reflex did not recover in animals implanted with 3T3 or C6 glioma cells or with vehicle only.

Topics: 3T3 Cells; Animals; Carbocyanines; Cells, Cultured; Female; Fluorescent Dyes; Glial Fibrillary Acidic Protein; Immunohistochemistry; Intestines; Mice; Muscle Contraction; Neuroglia; Rats; Rats, Wistar; Recovery of Function; Skin; Spinal Cord Injuries; Spinal Nerve Roots; Time Factors; Transplantation; Tumor Cells, Cultured

This research has examined the relationship between axonal regeneration and the return of normal movement following complete transection of the spinal cord. We made measurements of tail beat frequency and amplitude of the caudal body wave from video recordings of eels (Anguilla anguilla) swimming in a water tunnel at several speeds. Each eel was then anaesthetised and the spinal cord cut caudal to the anus; in some animals the resulting gap was filled with a rubber block. All animals were kept at 25 degrees C for recovery periods ranging from 7 to 128 days, during which their swimming performance was monitored regularly. Each fish was then re-anaesthetised and perfused with fixative and the regrowing descending axons labelled with 1,1'-diotadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate. For all animals and at all speeds after surgery, tail beat frequency increased, while amplitude decreased. In non-blocked animals, an improvement in performance was first seen from 8 days following transection and thereafter tail beat frequency decreased progressively until it had returned to normal after 35 to 45 days, while amplitude remained below baseline until at least 45 days. In these animals, few axonal growth cones had penetrated the caudal stump by 7 days, but some had extended as much as 3 mm by 15 days. Many had reached as far as 6 mm between 25 and 36 days, while by 128 days they had progressed up to 10.5 mm. Contralateral crossing was never observed. Functional recovery was never witnessed in animals in which the cord had been blocked and these eels swam at all times with elevated tail beat frequency and reduced caudal amplitude. No labelled axons could be traced into the caudal spinal cord at any recovery stage in such animals. We conclude that re-innervation of only 1-2 segments caudal to the injury is necessary for functional recovery, although continued axonal growth may be important for the refinement of some aspects of movement.

Topics: Anguilla; Animals; Carbocyanines; Denervation; Disease Models, Animal; Efferent Pathways; Fluorescent Dyes; Growth Cones; Locomotion; Movement Disorders; Nerve Regeneration; Recovery of Function; Spinal Cord; Spinal Cord Injuries; Swimming

This laboratory has demonstrated that lipid-coated microbubbles (LCMs) effectively aggregate and deliver chemotherapeutic drugs into rat brain tumor cells and antigliosis agents into maturing rat brain injury sites. In this study, we report the affinity of tail vein-injected LCMs to the injured rat spinal cord by a compressive lesion to the upper thoracic region.. The accumulation of LCMs in the injured spinal cord was analyzed by labeling it with a lipid-soluble fluorescent dye, 3,3'-dioctadecyloxacarbocyanine perchlorate. Indices of glial fibrillary acidic protein were measured concomitantly with 3,3'-dioctadecyloxacarbocyanine perchlorate-labeled LCMs using confocal microscopy.. There was no aggregation of LCMs accumulated 1 and 6 hours after injury; however, when given 2, 4, and 7 days after injury, LCMs showed a clear affinity for the injured region. LCM aggregation shifted from the central necrotic area of the injury on postinjury Day 2 and postinjury Day 4 to the white matter among glial fibrillary acidic protein-positive astrocytes by postinjury Day 7.. Affinity of LCMs for spinal cord injury sites may be mediated in the early stages after injury by proliferating macrophages in the necrotic center, and then in later stages by glial fibrillary acidic protein-positive astrocytes in adjacent white matter. These findings suggest a potential for using LCMs as a delivery vehicle to concentrate lipid-soluble agents in spinal cord injury sites.

Topics: Animals; Astrocytes; Carbocyanines; Fluorescent Antibody Technique; Fluorescent Dyes; Glial Fibrillary Acidic Protein; Injections, Intravenous; Lipids; Microscopy, Fluorescence; Microspheres; Necrosis; Rats; Rats, Sprague-Dawley; Spinal Cord; Spinal Cord Injuries; Surface Properties; Tail; Thorax; Time Factors; Wounds, Nonpenetrating

1. Repair and recovery following spinal cord injury (complete spinal cord crush) has been studied in vitro in neonatal opossum (Monodelphis domestica), fetal rat and in vivo in neonatal opossum. 2. Crush injury of the cultured spinal cord of isolated entire central nervous system (CNS) of neonatal opossum (P4-10) or fetal rats (E15-E16) was followed by profuse growth of fibres and recovery of conduction of impulses through the crush. Previous studies of injured immature mammalian spinal cord have described fibre growth occurring only around the lesion, unless implanted with fetal CNS. 3. The period during which successful growth occurred in response to a crush is developmentally regulated. No such growth was obtained after P12 in spinal cords crushed in vitro at the level of C7-8. 4. In vivo, in the neonatal (P4-8) marsupial opossum, growth of fibres through, and restoration of, impulse conduction across the crush was apparent 1-2 weeks after injury. With longer periods of time after crushing a considerable degree of normal locomotor function developed. 5. By the time the operated animals reached adulthood, the morphological structure of the spinal cord, both in the region of the crush and on either side of the site of the lesion, appeared grossly normal. 6. The results are discussed in relation to the eventual longterm possibility of devising effective treatments for patients with spinal cord injuries.

Topics: Animals; Animals, Newborn; Behavior, Animal; Carbocyanines; Electrophysiology; Female; Fluorescent Dyes; Immunohistochemistry; Microscopy, Electron; Neural Conduction; Neurons; Opossums; Pregnancy; Spinal Cord; Spinal Cord Injuries

The ability of neurites to grow through a lesion and form synaptic connections has been analyzed in a developing mammalian spinal cord in vitro. After isolation of the entire central nervous system (CNS) of the newly born South American opossum (Monodelphis domestica) the spinal cord was crushed. Outgrowth through and beyond the lesion was observed in living preparations for 2-5 days by staining axons with carbocyanine dyes. The structure of the acute crush and the growing neurites was examined by light and electron microscopy in tissue fixed immediately after the crush had been made. All axons had been severed and the site was filled with debris and amorphous vesicular structures. By 3 days after injury, numerous labelled neurites had grown into the lesion; by 4 days, many had extended several millimetres beyond it. At this time normal axonal profiles were apparent in electron micrographs of the crush site. Although fewer axons grew across the lesion than had been severed by the crush, the amplitudes of compound action potential volleys conducted across the crush in injured preparations were comparable with those recorded from uninjured spinal cords. Physiological experiments made with raised concentrations of extracellular magnesium in the culture fluid indicated that growing axons had formed synaptic connections. Thus, delayed major peaks of the response were abolished while the small component corresponding to through conduction remained unaffected by magnesium. These experiments demonstrate the development of synaptic interactions by the growing neurites and confirm the far greater powers of repair in neonatal mammals compared to adults. They set the stage for comparing molecular mechanisms involved in development and regeneration of the mammalian CNS.

Topics: Action Potentials; Animals; Animals, Newborn; Axons; Carbocyanines; Culture Techniques; Electrophysiology; Fluorescent Dyes; Magnesium; Microscopy, Electron; Nerve Crush; Opossums; Spinal Cord; Spinal Cord Injuries; Synapses