Computational molecular phenotyping of retinal sheet transplants to rats with retinal degeneration.

Retinal progenitor sheet transplants have been shown to extend neuronal processes into a degenerating host retina and to restore visual responses in the brain. The aim of this study was to identify cells involved in transplant signals to retinal degenerate hosts using computational molecular phenotyping (CMP). S334ter line 3 rats received fetal retinal sheet transplants at the age of 24-40 days. Donor tissues were incubated with slow-releasing microspheres containing brain-derived neurotrophic factor or glial cell-derived neurotrophic factor. Up to 265 days after surgery, eyes of selected rats were vibratome-sectioned through the transplant area (some slices stained for donor marker human placental alkaline phosphatase), dehydrated and embedded in Eponate, sectioned into serial ultrathin datasets and probed for rhodopsin, cone opsin, CRALBP (cellular retinaldehyde binding protein), l-glutamate, l-glutamine, glutathione, glycine, taurine, γ-aminobutyric acid (GABA) and DAPI (4',6-diamidino-2-phenylindole). In large transplant areas, photoreceptor outer segments in contact with host retinal pigment epithelium revealed rod and cone opsin immunoreactivity whereas no such staining was found in the degenerate host retina. Transplant photoreceptor layers contained high taurine levels. Glutamate levels in the transplants were higher than in the host retina whereas GABA levels were similar. The transplant inner nuclear layer showed some loss of neurons, but amacrine cells and horizontal cells were not reduced. In many areas, glial hypertrophy between the host and transplant was absent and host and transplant neuropil appeared to intermingle. CMP data indicate that horizontal cells and both glycinergic and GABAergic amacrine cells are involved in a novel circuit between transplant and host, generating alternative signal pathways between transplant and degenerating host retina.

In vitro differentiation potential of human embryonic versus adult stem cells.

BACKGROUND: There is widespread controversy regarding the potential of human neural stem cells and human mesenchymal stem cells (hMSCs) to form cell types outside of their normal developmental lineage. A greater understanding of the differentiation potential and bias of these stem cell types would allow researchers to select the cell type that best suits the research or clinical need at hand. MATERIALS & METHODS: We used identical in vitro protocols to quantitatively compare the potential of human embryonic stem cells, human neural stem cells and hMSCs to differentiate into specific ectodermal or mesodermal lineages. RESULTS: Our findings demonstrate that human embryonic stem cells and human neural stem cells have the ability to differentiate into high purity neuronal progenitor or oligodendrocyte progenitor cultures. By contrast, hMSCs generated exceedingly limited numbers of neural lineages. Both human embryonic stem cells and hMSCs generated adipocytes and osteocytes when exposed to mesodermal differentiation conditions. CONCLUSION: These studies underscore the importance of distinguishing differentiation potential from differentiation bias, an important consideration in the development of cell replacement strategies.

Visual restoration and transplant connectivity in degenerate rats implanted with retinal progenitor sheets.

The aim of this study was to determine whether retinal progenitor layer transplants form synaptic connections with the host and restore vision. Donor retinal sheets, isolated from embryonic day 19 rat fetuses expressing human placental alkaline phosphatase (hPAP), were transplanted to the subretinal space of 18 S334ter-3 rats with fast retinal degeneration at the age of 0.8-1.3 months. Recipients were killed at the age of 1.6-11.8 months. Frozen sections were analysed by confocal immunohistochemistry for the donor cell label hPAP and synaptic markers. Vibratome slices were stained for hPAP, and processed for electron microscopy. Visual responses were recorded by electrophysiology from the superior colliculus (SC) in 12 rats at the age of 5.3-11.8 months. All recorded transplanted rats had restored or preserved visual responses in the SC corresponding to the transplant location in the retina, with thresholds between -2.8 and -3.4 log cd/m(2). No such responses were found in age-matched S334ter-3 rats without transplants, or in those with sham surgery. Donor cells and processes were identified in the host by light and electron microscopy. Transplant processes penetrated the inner host retina in spite of occasional glial barriers between transplant and host. Labeled neuronal processes were found in the host inner plexiform layer, and formed apparent synapses with unlabeled cells, presumably of host origin. In conclusion, synaptic connections between graft and host cells, together with visual responses from corresponding locations in the brain, support the hypothesis that functional connections develop following transplantation of retinal layers into rodent models of retinal degeneration.

Neutralization of the chemokine CXCL10 reduces inflammatory cell invasion and demyelination and improves neurological function in a viral model of multiple sclerosis.

Intracerebral infection of mice with mouse hepatitis virus (MHV) results in an acute encephalomyelitis followed by a chronic demyelinating disease with clinical and histological similarities with the human demyelinating disease multiple sclerosis (MS). Following MHV infection, chemokines including CXC chemokine ligand (CXCL)10 (IFN inducible protein 10 kDa), CXCL9 (monokine induced by IFN-gamma), and CC chemokine ligand 5 (RANTES) are expressed during both acute and chronic stages of disease suggesting a role for these molecules in disease exacerbation. Previous studies have shown that during the acute phase of infection, T lymphocytes are recruited into the CNS by the chemokines CXCL10 and CXCL9. In the present study, MHV-infected mice with established demyelination were treated with antisera against these two chemokines, and disease severity was assessed. Treatment with anti-CXCL10 reduced CD4+ T lymphocyte and macrophage invasion, diminished expression of IFN-gamma and CC chemokine ligand 5, inhibited progression of demyelination, and increased remyelination. Anti-CXCL10 treatment also resulted in an impediment of clinical disease progression that was characterized by a dramatic improvement in neurological function. Treatment with antisera against CXCL9 was without effect, demonstrating a critical role for CXCL10 in inflammatory demyelination in this model. These findings document a novel therapeutic strategy using Ab-mediated neutralization of a key chemokine as a possible treatment for chronic human inflammatory demyelinating diseases such as MS.

Depletion of endogenous oligodendrocyte progenitors rather than increased availability of survival factors is a likely explanation for enhanced survival of transplanted oligodendrocyte progenitors in X-irradiated compared to normal CNS.

Oligodendrocyte progenitors (OPs) survive and migrate following transplantation into adult rat central nervous system (CNS) exposed to high levels of X-irradiation but fail to do so if they are transplanted into normal adult rat CNS. In the context of developing OP transplantation as a potential therapy for repairing demyelinating diseases it is clearly of some importance to understand what changes have occurred in X-irradiated CNS that permit OP survival. This study addressed two alternative hypotheses. Firstly, X-irradiation causes an increase in the availability of OP survival factors, allowing the CNS to support a greater number of progenitors. Secondly, X-irradiation depletes the endogenous OP population thereby providing vacant niches that can be occupied by transplanted OPs. In situ hybridization was used to examine whether X-irradiation causes an increase in mRNA expression of five known OP survival factors, CNTF, IGF-I, PDGF-A, NT-3 and GGF-2. The levels of expression of these factors at 4 and 10 days following exposure of the adult rat spinal cord to X-irradiation remain the same as the expression levels in normal tissue. Using intravenous injection of horseradish peroxidase, no evidence was found of X-irradiation-induced change in blood-brain barrier permeability that might have exposed X-irradiated tissue to serum-derived survival factors. However, in support of the second hypothesis, a profound X-irradiation-induced decrease in the number of OPs was noted. These data suggest that the increased survival of transplanted OPs in X-irradiated CNS is not a result of the increases in the availability of the OP survival factors examined in this study but rather the depletion of endogenous OPs creating 'space' for transplanted OPs to integrate into the host tissue.

Stem cell transplantation into the central nervous system and the control of differentiation.

Recent advances in stem cell biology, including methods of cell amplification and control of differentiation in vitro, provide us with new and powerful tools with which to explore the cellular, molecular, and genetic factors affecting cell survival, proliferation, differentiation, and differentiation potential. Mitigating this vein of enthusiasm are the results of stem cell transplantation studies, which highlight our inability to control the fate of stem cell populations following transplantation to the central nervous system (CNS). Differentiation of transplanted cells is strongly influenced by the environmental signals and cellular deficiencies operating at the site of implantation, over which we can exert little or no control. Where stem cell transplantation-mediated repair of the injured CNS has been demonstrated most successfully, the transplant environments have invariably been simplistic, and transplantation into the complex and reactive environment of a CNS injury site generally results in migration from the site of implantation followed by glial cell differentiation. Together, these findings suggest that the most significant advances for the stem cell transplantation field will come from research strategies that include predifferentiation of stem cells prior to transplant and studies that further our understanding of the factors affecting stem cell differentiation in the complex environment of the CNS in vivo.

Enhanced axonal regeneration following combined demyelination plus schwann cell transplantation therapy in the injured adult spinal cord.

We have treated spinal cord injured rats with demyelination plus Schwann cell transplantation and assessed neurite outgrowth in a quantifiable model of axonal regeneration. Axonal injuries of differing severity were induced in the dorsal funiculus of adult rats using a micromanipulator-controlled Scouten knife. Demyelinated regions were produced so as to overlap with the injury site by the injection of galactocerebroside antibodies plus complement one segment cranial to the axonal injury site. Schwann cells were isolated from the sciatic nerve, expanded in vitro, and transplanted into the injury site 1 day later. Animals were killed after an additional 7 days. Schwann cells were evenly distributed throughout the region of demyelination, which extended 6-7 mm cranial to the axonal injury site. The severity of axonal injury was quantified by counting degenerate axons in transverse resin sections. The degree of axonal regeneration was assessed by an electron microscopic analysis of growth cone frequency and distribution relative to the site of axonal injury. Quantification of growth cones at a distance from the site of axonal injury indicated a strong linear relationship (P < 0.001) between the number of growth cones and the number of severed axons; the ratio of growth cones to severed axons was increased by 26.5% in demyelinated plus transplanted animals compared to demyelinated animals without a transplant. Furthermore, only the demyelinated plus transplanted animals contained growth cones associated with myelin in white matter immediately outside of the region of complete demyelination. Growth cones were absent in transplanted-only animals at a distance from the site of axonal injury. These findings indicate that combined demyelination plus Schwann cell transplantation therapy enhances axonal regeneration following injury and suggests that growth cones are able to overcome myelin-associated inhibitors of neurite outgrowth in the presence of trophic support.

Polysialylated neural cell adhesion molecule-positive CNS precursors generate both oligodendrocytes and Schwann cells to remyelinate the CNS after transplantation.

Transplantation offers a means of identifying the differentiation and myelination potential of early neural precursors, features relevant to myelin regeneration in demyelinating diseases. In the postnatal rat brain, precursor cells expressing the polysialylated (PSA) form of the neural cell adhesion molecule NCAM have been shown to generate mostly oligodendrocytes and astrocytes in vitro (Ben-Hur et al., 1998). Immunoselected PSA-NCAM+ newborn rat CNS precursors were expanded as clusters with FGF2 and grafted into a focal demyelinating lesion in adult rat spinal cord. We show that these neural precursors can completely remyelinate such CNS lesions. While PSA-NCAM+ precursor clusters contain rare P75+ putative neural crest precursors, they do not generate Schwann cells in vitro even in the presence of glial growth factor. Yet they generate oligodendrocytes, astrocytes, and Schwann cells in vivo when confronted with demyelinated axons in a glia-free area. We confirmed the transplant origin of these Schwann cells using Y chromosome in situ hybridization and immunostaining for the peripheral myelin protein P0 of tissue from female rats that had been grafted with male cell clusters. The number and distribution of Schwann cells within remyelinated tissue, and the absence of P0 mRNAs in donor cells, indicated that Schwann cells were generated by expansion and differentiation of transplanted PSA-NCAM+ neural precursors and were not derived from contaminating Schwann cells. Thus, transplantation into demyelinated CNS tissue reveals an unexpected differentiation potential of a neural precursor, resulting in remyelination of CNS axons by PNS and CNS myelin-forming cells.

The origin of remyelinating cells in the central nervous system.

A clear understanding of the cellular events underlying successful remyelination of demyelinating lesions is a necessary prerequisite for an understanding of the failure of remyelination in multiple sclerosis (MS). The potential for remyelination of the adult central nervous system (CNS) has been well-established. However, there is still some dispute whether remyelinating oligodendrocytes arise from dedifferentiation and/or proliferation of mature oligodendrocytes, or are generated solely from proliferation and differentiation of glial progenitor cells. This review focuses on studies carried out on remyelinating lesions in the adult rat spinal cord produced by injection of antibodies to galactocerebroside and serum complement that show: (1) oligodendrocytes which survive within an area of demyelination do not contribute to remyelination, (2) remyelination is carried out by oligodendrocyte progenitor cells, (3) recruitment of oligodendrocyte progenitors to an area of demyelination is a local response, and (4) division of oligodendrocyte progenitors is symmetrical, resulting in chronic depletion of the oligodendrocyte progenitor population in the normal white matter around an area of remyelination. Such results suggest that repeated episodes of demyelination could lead to a failure of remyelination due to a depletion of oligodendrocyte progenitors.

The role of oligodendrocytes and oligodendrocyte progenitors in CNS remyelination.

Remyelination enables restoration of saltatory conduction and a return of normal function lost during demyelination. Unfortunately, remyelination is often incomplete in the adult human central nervous system (CNS) and this failure of remyelination is one of the main reasons for clinical deficits in demyelinating disease. An understanding of the failure of remyelination in demyelinating diseases such as Multiple Sclerosis depends upon the elucidation of cellular events underlying successful remyelination. Although the potential for remyelination of the adult CNS has been well established, there is still some dispute regarding the origin of the remyelinating cell population. The literature variously reports that remyelinating oligodendrocytes arise from dedifferentiation and/or proliferation of mature oligodendrocytes, or are generated solely from proliferation and differentiation of glial progenitor cells. This review focuses on studies carried out on remyelinating lesions in the adult rat spinal cord produced by injection of antibodies to galactocerebroside plus serum complement that demonstrate: 1) oligodendrocytes which survive within an area of demyelination do not contribute to remyelination, 2) remyelination is carried out by oligodendrocyte progenitor cells, 3) recruitment of oligodendrocyte progenitors to an area of demyelination is a local response, and 4) division of oligodendrocyte progenitors is symmetrical and results in chronic depletion of the oligodendrocyte progenitor population in the normal white matter around an area of remyelination. These results suggest that failure of remyelination may be contributed to by a depletion of oligodendrocyte progenitors especially following repeated episodes of demyelination. Remyelination allows the return of saltatory conduction (Smith et al., 1979) and the functional recovery of demyelination-induced deficits (Jeffery et al., 1997). Findings such as these have encouraged research aimed at enhancing the limited remyelination found in Multiple Sclerosis (MS) lesions, evidenced by a rim of thin myelin sheaths around the edges of a lesion, or, in a minority of acute foci, throughout the entire lesion (Prineas et al., 1989; Raine et al., 1981). It must be said, however, that although remyelination is clearly a prerequisite to sustained functional recovery, other factors such as the state of the inflammatory response and degree of axonal survival within the demyelinated region contribute to the extent of functional recovery that may be possible following therapeutic intervention aimed at halting disease progression. It is not yet clear whether the progression of functional deficits in MS is primarily the result of an increasing load of demyelination, or axon loss, or a combination of the two processes. However, given the increasing recognition that myelin sheaths play a role in protecting axons from degeneration, the success or failure of remyelination has functional consequences for the patient. To understand why remyelination should fail in demyelinating disease and develop strategies to enhance remyelination requires an understanding of the biology of successful remyelination. Firstly, what is the origin of the remyelinating cell population in the adult CNS? Secondly, what are the dynamics of the cellular response of this population during demyelination and remyelination? And thirdly, what are the consequences to the tissue of an episode of demyelination? This review will focus on studies that address these issues, and discuss the implications of the results of these experiments for our understanding of MS and the development of therapeutic interventions aimed at enhancing remyelination.

A quantifiable model of axonal regeneration in the demyelinated adult rat spinal cord.

Strategies to increase the extent of axonal regeneration in the adult CNS must address an array of intrinsic and environmental factors which influence neuritic outgrowth. In order to develop an in vivo model of axonal regeneration in which potential therapies may be assessed, we have quantified growth cones within demyelinated regions in the dorsal funiculus of the spinal cord, following a discrete axotomy. Demyelinated lesions were produced by the intraspinal injection of galactocerebroside antibodies plus serum complement proteins. Axonal integrity was not compromised by the demyelination protocol. Axonal injury was induced at the caudal extent of the demyelinated region using a micromanipulator-controlled Scouten knife. The severity of axonal injury was varied in different animals at the time of surgery and was quantified 8 days later by counting degenerate axons in transverse 1-microm resin sections. Evidence of axonal regeneration within these animals was assessed by an electron microscopic analysis of growth cone frequency and position relative to the site of axotomy. Growth cones were identified within the region of demyelination only; no growth cones were identified within the dorsal column white matter adjacent to the demyelinated region, or rostral or caudal to the region of demyelination, or in animals with an injury but no demyelination. Quantification of growth cones within regions of demyelination indicated a strong linear relationship (P < 0.001) between the number of growth cones and the number of axons severed. These findings indicate that demyelination facilitates axonal regeneration in the adult rat CNS and illustrate a quantifiable method of assessing axonal regeneration.

Response of the oligodendrocyte progenitor cell population (defined by NG2 labelling) to demyelination of the adult spinal cord.

Elucidation of the response of oligodendrocyte progenitor cell populations to demyelination in the adult central nervous system (CNS) is critical to understanding why remyelination fails in multiple sclerosis. Using the anti-NG2 monoclonal antibody to identify oligodendrocyte progenitor cells, we have documented their response to antibody-induced demyelination in the dorsal column of the adult rat spinal cord. The number of NG2+ cells in the vicinity of demyelinated lesions increased by 72% over the course of 3 days following the onset of demyelination. This increase in NG2+ cell numbers did not reflect a nonspecific staining of reactive cells, as GFAP, OX-42, and Rip antibodies did not co-localise with NG2 + cells in double immunostained tissue sections. NG2 + cells incorporated BrdU 48-72 h following the onset of demyelination. After the onset of remyelination (10-14 days), the number of NG2+ cells decreased to 46% of control levels and remained consistently low for 2 months. When spinal cords were exposed to 40 Grays of x-irradiation prior to demyelination, the number of NG2+ cells decreased to 48% of control levels by 3 days following the onset of demyelination and remained unchanged at 3 weeks. Since 40 Grays of x-irradiation kills dividing cells, these studies illustrate a responsive and nonresponsive NG2+ cell population following demyelination in the adult spinal cord and suggest that the responsive NG2+ cell population does not renew itself.

Identification of post-mitotic oligodendrocytes incapable of remyelination within the demyelinated adult spinal cord.

In order to investigate the remyelinating potential of mature oligodendrocytes in vivo, we have developed a model of demyelination in the adult rat spinal cord in which some oligodendrocytes survive demyelination. A single intraspinal injection of complement proteins plus antibodies to galactocerebroside (the major myelin sphingolipid) resulted in demyelination followed by oligodendrocyte remyelination. Remyelination was absent when the spinal cord was exposed to 40 Grays of x-irradiation prior to demyelination, a procedure that kills dividing cells. Quantitative Rip immunohistochemical analysis revealed a similar density of surviving oligodendrocytes in x-irradiated and nonirradiated lesions 3 days after demyelination. Rip and bromodeoxyuridine double immunohistochemical analysis of demyelinated lesions indicated that Rip+ oligodendrocytes did not divide as an acute response to demyelination. Oligodendrocytes were also identified by Rip immunostaining and electron microscopy at late time points (3 weeks) within x-irradiated areas of demyelination. These oligodendrocytes extended processes that engaged axons, and on occasion formed myelin membranes, but did not lay down new myelin sheaths. These studies demonstrate that (a) oligodendrocytes that survive within a region of demyelination are not induced to divide in the presence of demyelinated axons, and (b) fully-differentiated oligodendrocytes are therefore postmitotic and do not contribute to remyelination in the adult CNS.

In vivo immunological suppression of spinal cord myelin development.

The onset of myelination in the embryonic chick spinal cord begins on embryonic day (E) 12 or E13 of the 21 day in ovo developmental period. This event coincides with a loss of functional axonal regeneration following complete transection of the thoracic spinal cord. In this study, we have characterised an immunological method for delaying the developmental onset of myelination in vivo until later stages of development (developmental myelin-suppression). A single injection of heterologous or homologous serum complement proteins plus myelin-specific, complement-binding antibodies into the spinal cord prior to E13 delayed the onset of myelination until E17. The state of spinal cord myelin was assessed with immunohistochemical, histological and ultrastructural techniques. Northern blot analysis indicated that myelin basic protein mRNA was not down-regulated in myelin-suppressed spinal cords, which suggests that oligodendrocytes survived developmental myelin-suppression. Glial fibrillary acidic protein immunostaining of normal and treated tissue indicated that myelin-suppression did not alter the resident astrocyte population of the spinal cord or elicit astrogliosis. Immunostaining with microtubule-associated protein-2 and thionine staining of normal and myelin-suppressed tissue further indicated that the neuronal architecture was unaffected by the immunological protocol.

Axonal regeneration and physiological activity following transection and immunological disruption of myelin within the hatchling chick spinal cord.

Transections of the chicken spinal cord after the developmental onset of myelination at embryonic day (E) 13 results in little or no functional regeneration. However, intraspinal injection of serum complement proteins with complement-binding GalC or 04 antibodies between E9-E12 results in a delay of the onset of myelination until E17. A subsequent transection of the spinal cord as late as E15 (i.e., during the normal restrictive period for repair) results in neuroanatomical regeneration and functional recovery. Utilizing a similar immunological protocol, we evoked a transient alteration of myelin structure in the posthatching (P) chicken spinal cord, characterized by widespread "unravelling" of myelin sheaths and a loss of MBP immunoreactivity (myelin disruption). Myelin repair began within 7 d of cessation of the myelin disruption protocol. Long term disruption of thoracic spinal cord myelin was initiated after a P2-P10 thoracic transection and maintained for > 14 d by intra-spinal infusion of serum complement proteins plus complement-binding GalC or 04 antibodies. Fourteen to 28 d later, retrograde tract tracing experiments, including double-labeling protocols, indicated that approximately 6-19% of the brainstem-spinal projections had regenerated across the transection site to lumbar levels. Even though voluntary locomotion was not observed after recovery, focal electrical stimulation of identified brainstem locomotor regions evoked peripheral nerve activity in paralyzed preparations, as well as leg muscle activity patterns typical of stepping in unparalyzed animals. This indicated that a transient alteration of myelin structure in the injured adult avian spinal cord facilitated brainstem-spinal axonal regrowth resulting in functional synaptogenesis with target neurons.

Axonal regeneration contributes to repair of injured brainstem-spinal neurons in embryonic chick.

Recent results have demonstrated complete anatomical and functional repair of descending brainstem-spinal projections in chicken embryos that underwent thoracic spinal cord transection prior to embryonic day 13 (E13) of the 21 d developmental period. To determine to what extent axonal regeneration was contributing to this repair process, we conducted experiments using a double retrograde tract-tracing protocol. On E8-E13, the upper lumbar spinal cord was injected with the first fluorescent tracing dye to label those brainstem-spinal neurons projecting to the lumbar cord at that time. One to two days later (on E10-E15), the upper to mid-thoracic spinal cord was completely transected. After an additional 7-8 d, a different second fluorescent tracing dye was injected into the lumbar cord at least 5 mm caudal to the site of transection. Finally, 2 d later on E19 to postnatal day 4, the CNS was fixed and sectioned. Brainstem and spinal cord tissue sections were then viewed with epifluorescence microscopy. In comparison to nontrasected control animals, our findings indicated that there were relatively normal numbers of double-labeled brainstem-spinal neurons after a transection prior to E13, whereas the number of double-labeled and second-labeled brainstem-spinal neurons decreases after an E13-E15 transection. In addition, at each subsequent stage of development from E10 to E12, there was a greater number of double-labeled brainstem-spinal neurons (indicating regeneration of previously severed axons) than cell bodies labeled with the second fluorescent tracer alone (indicating subsequent development of late brainstem-spinal projections). Assessment of voluntary open-field locomotion (hatchling chicks) and/or brainstem-evoked locomotion (embryonic or hatchling) indicated that functional recovery of animals transected prior to E13 was indistinguishable from that observed in control chicks (sham operated or unoperated). Taken together, these data suggest that regeneration of previously axotomized fibers contributes to the observed anatomical and functional recovery after an embryonic spinal cord transection.

Suppression of the onset of myelination extends the permissive period for the functional repair of embryonic spinal cord.

In an embryonic chicken, transection of the thoracic spinal cord prior to embryonic day (E) 13 (of the 21-day developmental period) results in complete neuroanatomical repair and functional locomotor recovery. Conversely, repair rapidly diminishes following a transection on E13-E14 and is nonexistent after an E15 transection. The myelination of fiber tracts within the spinal cord also begins on E13, coincident with the transition from permissive to restrictive repair periods. The onset of myelination can be delayed (dysmyelination) until later in development by the direct injection into the thoracic cord on E9-E12 of a monoclonal antibody to galactocerebroside, plus homologous complement. In such a dysmyelinated embryo, a subsequent transection of the thoracic cord as late as E15 resulted in complete neuroanatomical repair and functional recovery (i.e., extended the permissive period for repair).

Functional repair of transected spinal cord in embryonic chick.

The purpose of this study was to determine the developmental stage of the chick embryo when descending spinal tracts lose the capacity for anatomical and functional repair after complete transection of the thoracic spinal cord. Previous studies have demonstrated that the first reticulospinal projections descend to the lumbar cord by embryonic day (E) 5. A comparison of the distribution and density of retrogradely labelled brainstem-spinal neurons in embryos versus hatchling chicks suggests that the descent of all brainstem-spinal projections is essentially complete to lumbar levels between E10 and El2. Transections and control sham operations were performed on different embryos from E3 through E14 of development. After a recovery period of 5-18 days, the extent of anatomical repair was assessed by injecting a small volume of a retrograde tract-tracing chemical into the upper lumbar spinal cord, caudal to the transection site. The brainstem nuclei were then examined for the number and distribution of retrogradely labelled brainstem-spinal neurons. In comparison to control animals, anatomical recovery appeared to be complete for embryos transected as late as E12, whereas thoracic cord transections conducted on E13-E14 resulted in reduced labelling of most brainstem-spinal nuclei. In addition, a number of E3-E6 transected embryos were allowed to hatch and with some assistance a few E7-E14 transected embryos also hatched. Functional recovery was assessed by behavioral observations and by focal electrical stimulation of brainstem locomotor regions (known to have direct projections to the lumbar spinal cord). Brainstem stimulation experiments were undertaken on transected and control embryos, either in ovo on E18-E20 or after hatching. Leg and wing muscle electromyographic recordings were used to monitor any brainstem evoked motor activity. Voluntary open-field locomotion (hatchling chicks) or brainstem evoked locomotion (embryonic or hatchling) in animals transected on or before E12 was indistinguishable from that observed in control (i.e. sham-operated or unoperated) chicks, indicating that complete functional recovery had occurred. In contrast, chicks transected on or after El3 showed reduced functional recovery. Since a previous study has shown that neurogenesis in chick brainstem-spinal neurons is complete prior to E5, the possible intrinsic neuronal mechanisms underlying the repair of descending supraspinal pathways are: (1) subsequent projections from later developing (undamaged) neurons, or (2) regrowth of previously axotomized projections (regeneration). For the E5-E12 chick embryos examined in this study, significant descending supraspinal fibers are present within the thoracic cord at the time of transection. Even if the transection is made at E12, when descending projections have completed their development to the lumbar cord, there is still a similar number and distribution of brainstem-spinal neurons labelled afterward (when compared to controls). This suggests that regeneration of previously axotomized projections may account for some of the observed anatomical and functional repair of brainstem-spinal pathways.