Search

The efficacy of Schwann cell-filled polycarbonate tubes as a bridging substrate for regrowing axons following lesions of the rat optic tract or cerebral cortex has been assessed after short (11-31 days) or long (82-119 days) survival times. Tubes were impregnated with Iaminin and poly-L-lysine, soaked in basic FGF and filled with Schwann cells. They were implanted into optic tract lesions in 34 rats aged 15-21 days and into cortical lesion cavities in 3 adult rats. Gelfoam soaked in basic FGF and Schwann cell conditioned medium was placed over the tubes. In one group of rats, axon regrowth into implants was assessed using neurofilament antibody RT97; antibodies to proteolipid protein, Po, Iaminin, the low-affinity nerve growth factor receptor (NGFr), S-100 and EDI were also used to study myelination and the cellular content of the tubes. In a second group of rats, anterograde tracing techniques were used to specifically identify host retinal axons within the implanted polymers. After long survival times, the relationships between regrown axons and cells inside the tubes were also examined ultrastructurally. In all implants examined immunohistochemically at short survival times, large numbers of RT97+ axons were found throughout the tubes, usually in association with Iaminin+, NGFr+ Schwann cells. At longer survival times, viable Schwann cells were still present, but tubes contained fewer axons and less cellular material. This material often formed a cord (200-250 μm thick) which extended the length of the implant. In the second group of rats, labelled retinal axons were found in 11 of the 16 implants that were attached to the dLGN. Axons regrew up to 1 mm but did not reach the distal (tectal) end of the implants. Interestingly, there was no evidence of myelinogenesis by either implanted Schwann cells or by host-derived oligodendroglia which had migrated into the tubes. Oligodendroglia were usually encircled by processes, many of which originated from Schwann cells, suggesting that the grafted cells may have been involved in isolating the central glia. The data show that Schwann cell-filled polycarbonate tubes provided a favourable milieu for axonal regeneration in the short term; however over time there was a decrease in the cellular and fibre content of the tubes. After intracranial implantation, an additional supporting matrix inside the polycarbonate tubes may aid in providing an environment conducive to the long term maintenance of regenerated retinal and other axons.

The use of nanoparticles for targeted delivery of therapeutic agents to sites of injury or disease in the central nervous system (CNS) holds great promise. However, the biodistribution of nanoparticles following in vivo administration is often unknown, and concerns have been raised regarding potential toxicity. Using poly(glycidyl methacrylate) (PGMA) nanoparticles coated with polyethylenimine (PEI) and containing superparamagnetic iron oxide nanoparticles as a magnetic resonance imaging (MRI) contrast agent and rhodamine B as a fluorophore, whole animal MRI and fluorescence analyses are used to demonstrate that these nanoparticles (NP) remain close to the site of injection into a partial injury of the optic nerve, a CNS white matter tract. In addition, some of these NP enter axons and are transported to parent neuronal somata. NP also remain in the eye following intravitreal injection, a non-injury model. Considerable infiltration of activated microglia/macrophages occurs in both models. Using magnetic concentration and fluorescence visualization of tissue homogenates, no dissemination of the NP into peripheral tissues is observed. Histopathological analysis reveals no toxicity in organs other than at the injection sites. Multifunctional nanoparticles may be a useful mechanism to deliver therapeutic agents to the injury site and somata of injured CNS neurons and thus may be of therapeutic value following brain or spinal cord trauma.

Polymer nanoparticles are widely used as a highly generalizable tool to entrap a range of different drugs for controlled or site-specific release. However, despite numerous studies examining the kinetics of controlled release, the biological behavior of such nanoparticles remains poorly understood, particularly with respect to endocytosis and intracellular trafficking. We synthesized polyethylenimine-decorated polymer nanospheres (ca. 100-250 nm) of the type commonly used for drug release and used correlated electron microscopy, fluorescence spectroscopy and microscopy, and relaxometry to track endocytosis in neural cells. These capabilities provide insight into how polyethylenimine mediates the entry of nanoparticles into neural cells and show that polymer nanosphere uptake involves three distinct steps, namely, plasma membrane attachment, fluid-phase as well as clathrin- and caveolin-independent endocytosis, and progressive accumulation in membrane-bound intracellular vesicles. These findings provide detailed insight into how the intracellular delivery of nanoparticles is mediated by polyethylenimine, which is presently the most commonly used nonviral gene transfer agent. This fundamental knowledge may also assist in the preparation of next-generation nonviral vectors.

Stabilization of enzymes has become a major focus in the quest to improve the activity, sustainability and recyclability of enzymes for their successful integration into both industry and medicine. Here, we describe the kinetic and thermodynamic stabilization of a variety of enzymes in the presence of cationic multifunctional polymeric nanoparticles.

The olfactory epithelium is a site of sustained adult neurogenesis where olfactory sensory neurons are continuously replaced from endogenous stem/progenitor cells. Epithelial macrophages have been implicated in the phagocytosis of degenerating cells but the molecular mechanisms allowing for their recruitment and activation while maintaining a neurogenic microenvironment are poorly understood. We have previously shown that the chemokine fractalkine (CX₃CL1) is expressed by olfactory sensory neurons and ensheathing cells in the olfactory epithelium. In turn, the fractalkine receptor, CX₃CR1, is expressed on macrophages and dendritic cells within the olfactory epithelium. We report that a selective cell death of olfactory sensory neurons in the epithelium of CX₃CR1-deficient mice via target ablation (i.e. olfactory bulbectomy) results in an exacerbated loss of olfactory sensory neurons compared to wild-type mice. In addition, reduced proliferation of intraepithelial stem/progenitor cells was observed in lesioned CX₃CR1-deficient mice, suggesting an impaired regenerative response. Importantly, a lack of CX₃CL1-signaling caused increased recruitment of macrophages into the olfactory epithelium, which in turn contained higher levels of pro-inflammatory cytokines (e.g. TNF-α and IL-6) as determined by qPCR. We also present novel data showing that, relative to wild-type, CX₃CR1-deficient macrophages have diminished phagocytic activity following stimulation with CX₃CL1. Collectively, our data indicate that signaling through the CX₃CR1 receptor modulates macrophage activity, resulting in an environment conducive to olfactory sensory neuron clearance and targeted replacement from endogenous stem/progenitor cells.

Attempts were made to enhance the regrowth of retinal axons which had been lesioned in the brachial region of the rat optic tract. Pieces of nitrocellulose paper (Millipore) were placed into the lesioned area between the dorsal lateral geniculate nucleus (dLGN) and superior colliculus (SC) in 13- to 18-day-old Wistar rats. Five types of implant were used: (1) uncoated implants, (2) coated with Poly-l-lysine (PLL), (3) coated on one side with cortical astrocytes, (4) coated with tectal astrocytes and (5) coated with Schwann cells. About half the Schwann cell-covered implants were precoated with PLL. Schwann cell-coated implants (16-40 × 103 cells per implant) were placed with the cells lying on either the dorsal or ventral (inverted) surface of the paper. 5-7 weeks after surgery, eyes were injected with WGA-HRP, the animals were perfused and frozen or vibratome sections (40-50 μm) processed for TMB histochemistry. Selected sections containing retinal axons were osmicated and prepared for electron microscopic examination. 45 out of 86 implants were found attached to the caudal dLGN. A small number of retinal axons were found growing onto the rostral end of one uncoated implant, two PLL-coated implants and over the surface of 4 of the astrocyte-coated implants. The densest and most extensive growth was seen on the Schwann cell-coated implants. In 15 of the 30 animals with such implants attached to the dLGN, retinal axons were found regrowing for 50-1120 μm (mean 530 μum). In about half of these rats (8 out of 15), the regrowth involved relatively large numbers of optic axons which were sometimes densely packed together. In the subgroup of Schwann cell-coated implants where the cells were placed upwards, retinal axons regrew on the dorsal surface of the paper for more than 500 μm in 7 animals. Almost no growth was seen on the uncoated (ventral) surface of the implants. In the subgroup of inverted implants, where the Schwann cells were placed downwards, in 4 animals retinal axon regrowth was densest on the ventral surface and extended for 300-550 μm. In 3 cases, occasional axons were also seen on the dorsal implant surface. Myelin sheaths surrounding some of the regenerating axons had PNS characteristics, suggesting that they were formed by the implanted Schwann cells, but most of the myelin appeared to be of central origin. The data suggest that Schwann cells placed just caudal to the dLGN and adjacent to lesioned retinal axons can enhance and direct the regrowth of these axons in the rat optic tract.

Recent clinical trials have shown that the use of replication deficient viral vectors to genetically modify cells in the retina can be of therapeutic benefit in the treatment of certain inherited degenerative conditions that compromise photoreceptor, and hence visual, function. This review is focussed primarily on the use of recombinant adeno-associated viral (rAAV) vectors to target neurons in inner retina, specifically retinal ganglion cells (RGCs). Genetic modification of RGCs may be of value in various ophthalmic conditions in which there is documented loss of RGCs or damage to their centrally projecting axons. Such conditions include glaucoma, optic neuritis, vascular disruption or trauma, and neurological degenerative conditions such as Alzheimer's disease. Furthermore, because the retina and optic nerve (ON) form part of the CNS, the visual system is a useful experimental model in which to study the molecular and cellular mechanisms that underlie degenerative as well as regenerative responses of adult CNS neurons after injury. Gene therapy studies from a number of laboratories are first reviewed, involving not only rAAV-based treatments but also application of lentiviral and adenoviral vectors. Recent work from our own laboratory is then summarized, in which intravitreal injection of rAAV2 serotype vectors is used to introduce growth promoting genes into injured RGCs. rAAV encoding a secretable form of ciliary neurotrophic factor (CNTF) has proved to be particularly effective in promoting RGC survival and axon regeneration after optic nerve crush or after transection followed by a peripheral nerve autograft. In the latter situation we have found that RGCs and their regenerated axons are maintained for at least 15 months after the initial injury. We have also combined rAAV gene therapy with pharmacotherapy to determine if cAMP elevation and additional intravitreal injections of growth factors can act synergistically with vector-based delivery of growth-promoting genes.

The extensive period of retinal ganglion cell (RGC) neurogenesis in the rat is associated with a protracted sequence of arrival of their axons into central targets such as the superior colliculus (SC) (Dallimore et al., 2002). Using in utero 5-bromo-2'-deoxyrudine (BrdU) injections to label early (embryonic day (E) 15) or late (E18 or E19) born RGCs, we now show that E15 RGCs with axons that enter the SC prenatally undergo programmed cell death earlier than late-born RGCs whose axons only reach the SC late in the first postnatal week. These late-born RGCs do not begin to die until postnatal day (P) 5/6. Removal of retrograde trophic support by P1 SC ablation initially only affects E15 RGCs; however by P5 death of late-born RGCs is increased, confirming that a switch to target dependency is delayed in this cohort. In a further experiment it was found that, following complete rostral SC transection at P2, the proportion of post-lesion axons originating from E19 RGCs was significantly greater than the proportion that normally makes up the retinotectal projection. Thus, even in neonatal brain, uninjured late-arriving axons are more likely to grow across a lesion site than injured axons undergoing regeneration. To study if birth date also affects regenerative potential in adulthood, autologous peripheral nerve (PN) was grafted onto the cut optic nerve in mature BrdU labelled rats. We found that, compared to E15 RGCs, a significantly greater proportion of late-born RGCs survived axotomy, but comparatively fewer of these surviving E19 RGCs regrew an axon into a graft. In summary, this research shows that the birthdate of RGCs significantly impacts on their subsequent life history and response to injury. Understanding how developing central nervous system (CNS) neurons acquire dependency on target-derived trophic support may lead to new strategies for enhancing survival and regeneration in adult CNS.

This study has examined the deleterious effect of superior colliculus (SC) ablation on the viability of identified retinotectally projecting ganglion cells in the neonatal rat retina. The time-course and extent of lesion-induced retinal ganglion cell (rgc) death has been determined and an estimate obtained for the rate of clearance of individual dying neurons. In order to demonstrate the projection of rgcs to the SC and the subsequent death of these same neurons after SC lesions, the fluorescent dye diamidino yellow (DY) was injected into the left SC of anesthetized 2 day old Wistar rats (P2: day of birth = P0). DY retrogradely labels the nuclei of tectally projecting rgcs; if these identified rgcs subsequently die, their DY-labelled nuclei become pyknotic and can be visualized in retinal wholemounts. At P4 the rats were again anesthetized and the injected area, seen as a yellow patch in the SC, was removed by aspiration. Rats were perfused 2 to 336 hours after the lesion and retinal wholemounts of the right eye were prepared. Control rats received only DY injections and were perfused at times corresponding to the lesioned animals. In three sham-operated rats; the injected SC was reexposed at P4 but the tectal tissue was not removed. In each of the 42 rats that were analyzed, about 10% of the retina containing retrogradely labelled rgcs was counted; the number of pyknotic versus normally labelled rgcs was determined and changes in normal cell density were also assessed. Pyknotic rates in control and sham-operated rats were similar (average O.8%, n = 11). In SC-lesioned rats, the proportion of pyknotic DY-labelled rgcs increased to about 2.5% 4 to 8 hours postlesion (PL); the peak period of death occurred at 23 hours PL (8.0%). The amount of pyknosis decreased thereafter and most dying cells had been eliminated by 50 hours PL. Phagocytosis of dying cells was a common feature of retinae in SC lesioned rats. In the long-term (336 hours) rats, counts of normal DY-labelled rgcs in corresponding regions of control and lesioned rats revealed an average decrease in rgc density of 47.3% after P4 tectal ablation. Calculations suggest a clearance time of about 3 hours for dying neonatal rgcs. © 1992 Wiley-Liss, Inc.

Secondary degeneration in the central nervous system involves indirect damage to neurons and glia away from the initial injury. Partial transection of the dorsal optic nerve (ON) results in precise spatial separation of the primary trauma from delayed degenerative events in ventrally placed axons and parent somata. Here we conduct an immunohistochemical survey of secondary cellular changes in and around axons and their parent retinal ganglion cell (RGC) somata during the first 3 days after a restricted, dorsal ON transection. This is before the secondary loss of RGCs and axons projecting through the uninjured, ventral portion of the ON. Within 5 min, manganese superoxide dismutase (MnSOD; a marker of oxidative stress) co-localizes within the astrocytic network across the entire profile of the ON. Secondary astrocyte hypertrophy of immunofluorescent labeling was evident from 3 h, with sustained increases in myelin basic protein immunoreactivity across the nerve by 24 h. Increases in NG-2-positive oligodendrocyte precursor cells, ED-1-positive activated microglia/macrophages, and Iba1-positive reactive resident microglia/macrophage numbers were only seen in ON vulnerable to secondary degeneration by 3 days. Changes within RGC somata exclusively vulnerable to secondary degeneration were detected at 24 h, as evidenced by increases in MnSOD immunoreactivity, followed by increases in c-jun immunoreactivity at 3 days. Treatment with the voltage-gated calcium channel blocker lomerizine did not alter any measured outcome. We conclude that oxidative stress spreading via the astrocytic network and from injured axons to parent RGC somata is an early event during secondary degeneration, and containment is likely to be required in order to prevent further damage to the nerve.

The mature CNS has limited intrinsic capacity for repair after injury; therefore, strategies are needed to enhance the viability and regrowth of damaged neurons. Here we review gene therapy studies in the eye, aimed at improving the survival and regeneration of injured retinal ganglion cells (RGCs). To target RGCs most current methods use recombinant adeno-associated viral vectors (AAV), usually serotype-2 (AAV2), that are injected into the vitreal chamber of the eye. This vector provides long-term transduction of adult RGCs. Strong, constitutive promoters such as CMV and/or beta-actin are commonly used but cell-specific promoters have also been tested. Transgenes encoded by AAV have been selected to limit cell death, enhance growth factor expression, or promote growth cone responsiveness. We have assessed the effects of AAV vectors in adult rodent models (i) after optic nerve (ON) crush and (ii) after transplantation of peripheral nerve (PN) onto the cut ON, a procedure that induces injured RGCs to regenerate axons over longer distances. AAV-CNTF-GFP promotes RGC survival and axonal regrowth in mice after ON crush, and in rats after ON crush or PN transplantation. In rats, intravitreal injection of AAV-BDNF-GFP also increases RGC viability but does not promote regeneration. RGC viability and axonal regrowth is further enhanced when AAV-CNTF-GFP is injected into transgenic mice that over-express bcl-2. Reconstituted PN grafts containing Schwann cells that were transduced ex vivo with lentiviral (LV) vectors encoding a secretable form of CNTF support RGC axonal regrowth, however grafts containing Schwann cells transduced with LV-BDNF or LV-GDNF are less successful. We have also quantified the transduction efficiency and tropism of different AAV vectors injected intravitreally. AAV 2/2 and AAV 2/6 showed highest levels of transduction, AAV 2/8 the lowest, and each serotype displayed different transduction profiles for retinal cells. We are also studying the long-term impact of AAV2-mediated CNTF or BDNF expression on the dendritic morphology of RGCs in normal and PN grafted retinas. Analysis of regenerating RGCs intracellularly injected with lucifer yellow indicates gene-specific changes in dendritic structure that likely impact upon visual function.

To assess the dynamics and confounding variables that influence transgene expression in neural stem cells (NSCs), we generated distinct NSC clones from the same pool of cells, carrying the same reporter gene transcribed from the same promoter, transduced by the same retroviral vector, and transplanted similarly at the same differentiation state, at the same time and location, into the brains of newborn mouse littermates, and monitored in parallel for over a year in vivo (without immunosuppression). Therefore, the sole variables were transgene chromosomal insertion site and copy number. We then adapted and optimized a technique that tests, at the single cell level, persistence of stem cell-mediated transgene expression in vivo based on correlating the presence of the transgene in a given NSC's nucleus (by fluorescence in situ hybridization [FISH]) with the frequency of that transgene's product within the same cell (by combined immunohistochemistry [IHC]). Under the above-stated conditions, insertion site is likely the most contributory variable dictating transgene downregulation in an NSC after 3 months in vivo. We also observed that this obstacle could be effectively and safely counteracted by simple serial infections (as few as three) inserting redundant copies of the transgene into the prospective donor NSC. (The preservation of normal growth control mechanisms and an absence of tumorigenic potential can be readily screened and ensured ex vivo prior to transplantation.) The combined FISH/IHC strategy employed here for monitoring the dynamics of transgene expression at the single cell level in vivo may be used for other types of therapeutic and housekeeping genes in endogenous and exogenous stem cells of many organs and lineages.

We here describe various approaches using GFP that are being used in the morphological and functional analysis of specific cell types in the normal and injured central nervous system. Incorporation of GFP into viral vectors allows phenotypic characterization of transduced cells and can be used to label their axons and terminal projections. Characterization of transduced cell morphology can be enhanced by intracellular injection of living GFP-labeled cells with appropriate fluorescent dyes. Ex vivo labeling of precursor or glial cells using viral vectors that encode GFP permits long-term identification of these cells after transplantation into the brain or spinal cord. In utero electroporation methods result in expression of gene products in developing animals, allowing both functional and morphological studies to be carried out. GFPCre has been developed as a marker gene for viral vector-mediated expression of the bacterial recombinase Cre in the brain of adult mice with "floxed" transgenes. GFPCre-mediated induction of transgene expression can be monitored by GFP expression in defined populations of neurons in the adult brain. Finally, GFP can be used to tag proteins, permitting dynamic visualization of the protein of interest in living cells.

The adult olfactory epithelium has maintained the ability to reconstitute its olfactory sensory neurons (OSNs) from a basal progenitor cell compartment. This allows for life-long turnover and replacement of receptor components as well as repair of the primary olfactory pathway in response to injury and environmental insults. The present study investigated whether fibulin-3, a glycoprotein in the extracellular matrix and binding partner of tissue inhibitor of metalloproteinases-3 (TIMP-3), plays a role in ongoing plasticity and regenerative events in the adult primary olfactory pathway. In wild-type control mice, fibulin-3 protein was detected on IB4(+)CD31(+) blood vessels, nerve fascicles and the basement membrane underneath the olfactory epithelium. After target ablation (olfactory bulbectomy), fibulin-3 was also abundantly present in the central nervous system (CNS) scar tissue that occupied the bulbar cavity. Using two different lesion models, i.e. intranasal Triton X-100 lesion and olfactory bulbectomy, we show that fibulin-3 deficient (Efemp1(-/-)) mice have impaired recovery of the olfactory epithelium after injury. Ten days post-injury, Efemp1(-/-) mice showed altered basal stem/progenitor cell proliferation and increased overall numbers of mature (olfactory marker protein (OMP) -positive) versus immature OSNs. However, compromised regenerative capacity of the primary olfactory pathway in Efemp1(-/-) mice was evidenced by reduced numbers of mature OSNs at the later time point of 42 days post-injury. In addition to these neural differences there were consistent changes in blood vessel structure in the olfactory lamina propria of Efemp1(-/-) mice. Overall, these data suggest a role for fibulin-3 in tissue maintenance and regeneration in the adult olfactory pathway.

We here describe various approaches using GFP that are being used in the morphological and functional analysis of specific cell types in the normal and injured central nervous system. Incorporation of GFP into viral vectors allows phenotypic characterization of transduced cells and can be used to label their axons and terminal projections. Characterization of transduced cell morphology can be enhanced by intracellular injection of living GFP-labeled cells with appropriate fluorescent dyes. Ex vivo labeling of precursor or glial cells using viral vectors that encode GFP permits long-term identification of these cells after transplantation into the brain or spinal cord. In utero electroporation methods result in expression of gene products in developing animals, allowing both functional and morphological studies to be carried out. GFPCre has been developed as a marker gene for viral vector-mediated expression of the bacterial recombinase Cre in the brain of adult mice with "floxed" transgenes. GFPCre-mediated induction of transgene expression can be monitored by GFP expression in defined populations of neurons in the adult brain. Finally, GFP can be used to tag proteins, permitting dynamic visualization of the protein of interest in living cells.

The mature CNS has limited intrinsic capacity for repair after injury; therefore, strategies are needed to enhance the viability and regrowth of damaged neurons. Here we review gene therapy studies in the eye, aimed at improving the survival and regeneration of injured retinal ganglion cells (RGCs). To target RGCs most current methods use recombinant adeno-associated viral vectors (AAV), usually serotype-2 (AAV2), that are injected into the vitreal chamber of the eye. This vector provides long-term transduction of adult RGCs. Strong, constitutive promoters such as CMV and/or beta-actin are commonly used but cell-specific promoters have also been tested. Transgenes encoded by AAV have been selected to limit cell death, enhance growth factor expression, or promote growth cone responsiveness. We have assessed the effects of AAV vectors in adult rodent models (i) after optic nerve (ON) crush and (ii) after transplantation of peripheral nerve (PN) onto the cut ON, a procedure that induces injured RGCs to regenerate axons over longer distances. AAV-CNTF-GFP promotes RGC survival and axonal regrowth in mice after ON crush, and in rats after ON crush or PN transplantation. In rats, intravitreal injection of AAV-BDNF-GFP also increases RGC viability but does not promote regeneration. RGC viability and axonal regrowth is further enhanced when AAV-CNTF-GFP is injected into transgenic mice that over-express bcl-2. Reconstituted PN grafts containing Schwann cells that were transduced ex vivo with lentiviral (LV) vectors encoding a secretable form of CNTF support RGC axonal regrowth, however grafts containing Schwann cells transduced with LV-BDNF or LV-GDNF are less successful. We have also quantified the transduction efficiency and tropism of different AAV vectors injected intravitreally. AAV 2/2 and AAV 2/6 showed highest levels of transduction, AAV 2/8 the lowest, and each serotype displayed different transduction profiles for retinal cells. We are also studying the long-term impact of AAV2-mediated CNTF or BDNF expression on the dendritic morphology of RGCs in normal and PN grafted retinas. Analysis of regenerating RGCs intracellularly injected with lucifer yellow indicates gene-specific changes in dendritic structure that likely impact upon visual function.

This chapter deals with projections from brain to spinal cord. 27 brain centers have been identified which send descending tracts to the spinal cord in mammals. The best known of these descending tracts is the corticospinal tract, which is a huge fiber bundle in humans and the other anthropoid primates. Apart from the corticospinal tract, which originates from a number of cortical areas, the most prominent descending tracts are those that arise from the red nucleus (rubrospinal), the vestibular nuclei (lateral and medial vestibulospinal), and the hindbrain reticular formation (lateral and medial reticulospinal). The other sites of origin of descending tracts, which are smaller in size, include the hypothalamus (paraventricular nucleus), prethalamus (zona incerta), pretectum (nucleus of Darkschewitsch), midbrain (superior colliculus, periaqueductal gray, supraoculomotor nucleus, interstitial nucleus of Cajal, and cuneiform nucleus), and rhombencephalon (parabigeminal nucleus, medial cerebellar nucleus, locus coeruleus, subcoeruleus nuclei, nucleus gigantocellularis, raphe, and the nucleus of the solitary tract). The origin and topography of supraspinal projections in humans is beginning to be elucidated using modern diffusion tensor imaging and tractography.

Publisher Summary The majority of spinal cord injury (SCI) events occur in the 18–35 year age group and the long-term personal, social and economic costs are enormous. Increasing use is being made of experimental animal models of SCI. The aims are to better understand the acute and chronic morphological, cellular and molecular consequences of SCI, and with this information develop and test new therapies to enhance anatomical repair and functional recovery. The ultimate aim is improved treatment and rehabilitation of patients suffering SCI. As the goal for clinical therapies becomes increasingly reachable, associating these often broad behavioral outcomes with experimental strategies aimed at tissue repair/sparing, remyelination, neuronal protection, cellular replacement, and axonal regeneration, as well as the activation of endogenous host stem/progenitor cell responses, has become more crucial but also more complex. There are many differences between the nervous systems of humans and animals commonly used in SCI studies. Issues such as size, gait, neuroanatomical, neurophysiological, and behavioral differences as well as disparities in immunological and inflammatory responses following SCI, all point to potential limitations of animal models when assessing the efficacy and safety of possible SCI treatments in humans. Use of non-human primates as an intermediate step between rodents and humans may aid in translation of effective therapies to the clinic.

Injured adult retinal ganglion cells (RGCs) regrow axons into peripheral nerve (PN) grafted onto cut optic nerve. Survival and regeneration of RGCs is increased by intraocular injections of ciliary neurotrophic factor (CNTF) and axonal regeneration is further enhanced by co-injection of a cyclic AMP analogue (CPT-cAMP). Based on these data, and because cytokine signaling is negatively regulated by suppressor of cytokine signaling (SOCS) proteins, we set out to determine whether CNTF injections increase retinal SOCS expression and whether any changes are attenuated by co-injection with CPT-cAMP. Using quantitative PCR we found increased SOCS1, SOCS2 and SOCS3 mRNA levels at various times after a single CNTF injection. Expression remained high for many days. SOCS protein levels were also increased. In situ hybridization revealed that RGCs express SOCS3 mRNA, and SOCS expression in cultured RGCs was increased by CNTF. Co-injection of CPT-cAMP reduced CNTF induced expression of SOCS1 and SOCS3 mRNA and decreased SOCS3 protein expression. CNTF injection also transiently increased retinal leukemia inhibitory factor (LIF) expression, an effect that was also moderated by CPT-cAMP. We propose that, along with known reparative effects of elevated cAMP on neurons, reducing SOCS upregulation may be an additional way in which cyclic nucleotides augment cytokine-induced regenerative responses in the injured CNS.

In order to promote neurite outgrowth, we constructed a plasmid producing RhoA-specific small hairpin RNA (shRNA) to block RhoA expression and tested its actions in PC12 cells. Our results show that the shRNA-mediated RNA interference (RNAi) successfully suppressed RhoA expression. As a consequence of RhoA knockdown, F-actin levels were significantly reduced and processes were markedly induced. These processes express two neurite markers, neurofilament and betaIII tubulin. This study demonstrates that plasmid-producing shRNA specific for RhoA can act as an effective tool to induce neurite outgrowth and further confirms the neurite growth-inhibitory role of RhoA. This shRNA may thus be a useful tool in promoting neurite outgrowth and could be applicable in neural repair after CNS injury.

Retrograde tracing techniques have been used to study the host visual cortical projection to fetal tectal tissue grafted to the midbrain of newborn host rats. To determine whether there is any topographic order in these cortical afferents, different parts of the grafts were injected with 3 different tracers: Fast blue (FB), Diamidino yellow dihydrochloride (DY), and either horseradish peroxidase (HRP) or rhodamine-labelled microspheres (Rh). The comparative visual cortical distribution of cells retrogradely labelled with the different dyes was then examined. Tectal tissue from 15-day-old pigmented rat embryos was injected via a glass micropipette onto the dorsal midbrain of anaesthetised newborn rats of the same strain. In adulthood, host rats were examined for the presence of grafts; 21 grafts were injected with retrograde tracers and the cortices of 12 of these animals were mapped to show the relative location of FB-, DY-, HRP- or Rh-labelled cells. Qualitative inspection of area 17 did not reveal consistent evidence of point-to-point visuotopic mapping in the cortico-transplant projection. However, within area 17 statistical analysis (chi 2 tests) revealed significant differences in most brains in the relative distribution of FB-, DY-, HRP- or Rh-labelled neurons. Areas 18 and 18a contained greater numbers of retrogradely labelled cells. In these extrastriate regions, statistical analysis also indicated significant differences in the relative distribution of neurons labelled with different tracers. These data thus provide evidence for a non-random pattern of cortical innervation of tectal grafts. Possible reasons for the absence of coherent, topographically organized cortico-transplant maps typical of the normal corticotectal projection are considered.

The rat occipital cortex contains a number of morphologically and physiologically distinct visual areas. Retrograde tracing studies have shown that most if not all of these areas project in a topographic fashion to the ipsilateral superior colliculus (SC). In the present study, small amounts of wheat germ agglutinin-conjugated HRP (WGA-HRP) were injected into adult rat occipital cortex to determine how afferents from the different visual cortical areas are distributed within the various layers of the SC. Cytoarchitectonic criteria were used to help establish the location of the WGA-HRP injections in the cortex. As a further aid to identifying the sites of injection, the distribution of retrogradely labelled cells within the thalamus was mapped in each brain. Analysis revealed a surprising range of visuocortical projections to the rat SC, with input to the majority of tectal laminae. Area 17 projected most heavily to the dorsal stratum opticum (SO) and lower half of stratum griseum superficiale (SGS) with lighter label extending up to the collicular surface. Axons and terminals from area 18 formed two horizontal tiers, one in the middle of the stratum griseum intermediale (SGI) and the other at the border between the stratum album intermediale (SAI) and the stratum griseum profundum (SGP). Periodic puffs of label extended between these horizontal tiers, with a periodicity of 300-400 microns. There was some variability in the labelling pattern in the superior colliculus after area 18a injections, perhaps because this cytoarchitectonic area contains multiple representations of the visual field. Generally the projection from the lateromedial, laterointermediate, and laterolateral parts of 18a was heaviest in the lower half of SO and upper regions of SGI. Lighter label extended up into the lower half of SGS. In the SGI it was common to find periodic puffs of terminal label interspersed with areas devoid of innervation. This periodic pattern was particularly noticeable after WGA-HRP injections into anterior 18a. Two horizontal tiers of label were located at the SO/SGI border and SGI/SAI border (the dorsal being the most dense) with patches of label extending between these tiers every 230-250 microns. There did not appear to be a significant corticotectal projection from the posterior part of the lateral extrastriate region.

The immune response after neural injury influences the survival and regenerative capacity of neurons. In the primary visual pathway, previous studies have described beneficial effects of macrophages and T-cells in promoting neural survival and axonal regeneration in some rat strains. However, the contributions of specific cell populations to these responses have been unclear. In adult Fischer (F344) rats, we confirm prior reports that intravitreal macrophage activation promotes the survival of retinal ganglion cells (RGCs) and greatly enhances axonal regeneration through a peripheral nerve graft. Neonatal thymectomy that results in elimination of T-cell production enhanced RGC survival after axotomy, but diminished the effect of intravitreal macrophage activation on axon regeneration. Thus, in F344 rats, lymphocytes appear to suppress RGC survival but augment the pro-regenerative effects of macrophages. The cytotoxic effect of lymphocytes on RGCs was confirmed in in vitro studies; coculture of retinal explants with lymphocytes led to a 60% reduction in viable RGCs. Similar in vivo results were obtained in Sprague Dawley rats. By comparison, in adult Lewis rats, neither RGC survival nor axonal regeneration was increased after intravitreal macrophage activation. Neonatal thymectomy had only a small beneficial effect on RGC survival, and although Lewis lymphocytes reduced RGC viability in culture, they did so to a lesser extent. Thus, in addition to a complex role of lymphocytes, particularly T-cells, after central nervous system injury, the present results demonstrate that the impact of macrophages is also influenced by genetic background.

In this Chapter we shall briefly review our recent studies in neonatal rats regarding the mechanisms involved in retinal ganglion cell death (RGC) after loss of central visual target areas in the brain. We also describe the influence of neurotrophins on RGC survival and consider the apparently symbiotic relationship between these retinal neurons and the cells they innervate in the developing brain.

We used a gene therapy approach in transgenic mice to assess the cooperative effects of combining anti-apoptotic and growth-promoting stimuli on adult retinal ganglion cell (RGC) survival and axonal regeneration following intraorbital optic nerve injury. Bi-cistronic adeno-associated viral vectors encoding a secretable form of ciliary neurotrophic factor and green fluorescent protein (AAV-CNTF-GFP) were injected into eyes of mice that had been engineered to over-express the anti-apoptotic protein bcl-2. For comparison this vector was also injected into wildtype (wt) mice, and both mouse strains were injected with control AAV encoding GFP. Five weeks after optic nerve injury we confirmed that bcl-2 over-expression by itself promoted the survival of axotomized RGCs, but in contrast to previous reports we also saw regeneration of some mature RGC axons beyond the optic nerve crush. AAV-mediated expression of CNTF in adult retinas significantly increased the survival and axonal regeneration of RGCs following axotomy in wt and bcl-2 transgenic mice; however, the effects were greatest in the transgenic strain. Compared with AAV-GFP-injected bcl-2 mice, RGC viability was increased by about 50% (mean, 36 738 RGCs per retina), and over 1000 axons per optic nerve regenerated 1-1.5 mm beyond the crush. These findings exemplify the importance of using a multifactorial therapeutic approach that enhances both neuroprotection and regeneration after central nervous system injury.

Similar to neurons in the peripheral nervous system, immature CNS-derived RGCs become dependent on target-derived neurotrophic support as their axons reach termination sites in the brain. To study the factors that influence this developmental transition we took advantage of the fact that rat RGCs are born, and target innervation occurs, over a protracted period of time. Early-born RGCs have axons in the SC by birth (P0), whereas axons from late-born RGCs do not innervate the SC until P4-P5. Birth dating RGCs using EdU allowed us to identify RGCs (1) with axons still growing toward targets, (2) transitioning to target dependence, and (3) entirely dependent on target-derived support. Using laser-capture microdissection we isolated ∼34,000 EdU+RGCs and analyzed transcript expression by custom qPCR array. Statistical analyses revealed a difference in gene expression profiles in actively growing RGCs compared with target-dependent RGCs, as well as in transitional versus target-dependent RGCs. Prior to innervation RGCs expressed high levels of BDNF and CNTFR α but lower levels of neurexin 1 mRNA. Analysis also revealed greater expression of transcripts for signaling molecules such as MAPK, Akt, CREB, and STAT. In a supportingin vitrostudy, purified birth-dated P1 RGCs were cultured for 24-48 h with or without BDNF; lack of BDNF resulted in significant loss of early-born but not late-born RGCs. In summary, we identified several important changes in RGC signaling that may form the basis for the switch from target independence to dependence.

The basic helix-loop-helix (bHLH) transcription factor Scl displays tissue-restricted expression and is critical for the establishment of the haematopoietic system; loss of Scl results in embryonic death due to absolute anaemia. Scl is also expressed in neurons of the mouse diencephalon, mesencephalon and metencephalon; however, its requirement in those sites remains to be determined. Here we report conditional deletion of Scl in neuronal precursor cells using the Cre/LoxP system. Neuronal-Scl deleted mice died prematurely, were growth retarded and exhibited an altered motor phenotype characterized by hyperactivity and circling. Moreover, ablation of Scl in the nervous system affected brain morphology with abnormal neuronal development in brain regions known to express Scl under normal circumstances; there was an almost complete absence of Scl-null neurons in the hindbrain and partial loss of Scl-null neurons in the thalamus and midbrain from early neurogenesis onwards. Our results demonstrate a crucial role for Scl in the development of Scl-expressing neurons, including gamma-aminobutyric acid (GABA)ergic interneurons. Our study represents one of the first demonstrations of functional overlap of a single bHLH protein that regulates neural and haematopoietic cell development. This finding underlines Scl's critical function in cell fate determination of mesodermal as well as neuroectodermal tissues.

In vivo, transplanted adult olfactory ensheathing glia (OEG) and adult Schwann cells (SC) can support the regrowth of at least some transected axons within adult CNS neuropil. In the present study, we developed an in vitro adult rat retinal explant model to explore the influence of primary adult SC and OEG on retinal ganglion cell (RGC) neurite regrowth in the presence of glial cells endogenous to the retina. Retinal quadrants were plated RGC-side down onto aclar hats coated with either pure collagen (type 1), collagen with OEG, collagen with SCs, or collagen coated with both OEG and SCs. Regrowing retinal neurites extended onto the pure collagen substrate, largely in association with astrocytes that migrated out from the explants (mean number of neurites: 144 ± 65 SEM). The additional presence of OEG (669 ± 122), but not SCs (97 ± 41), supported the regrowth of significantly greater numbers of RGC neurites. Furthermore, this OEG-stimulated regeneration was over significantly greater distances; >68% of neurites extended > 500 μm from the explant, compared with explants plated onto SCs or collagen alone (15% and 29%, respectively). When OEG and SCs were co-cultured the number of regenerating neurites was reduced (397 ± 81) compared with the pure OEG treatment. Analysis of explants on pure collagen substrates fed with media conditioned by purified OEG or SC showed no increase in neurite outgrowth compared with control treatments, suggesting that the enhanced growth in the presence of OEG is a contact-mediated effect. The observed differences between the abilities of OEG and SC to support the growth of CNS-derived fibers in the presence of astrocytes support the suggestion that OEG may be better suited for direct transplantation into CNS neuropil following injury. © 2005 Wiley-Liss, Inc.

Adenoviral (AdV) vectors encoding neurotrophin-3 (AdV-NT-3) or the bacterial marker enzyme beta-galactosidase (LacZ gene) were used to transduce olfactory ensheathing glia (OEG) cultures. AdV vector-transduced OEG expressed high levels of recombinant neurotrophin as shown by in situ hybridization and enzyme-linked immunosorbent assay techniques. The biological activity of vector-derived NT-3 was determined in a dorsal root ganglia neurite outgrowth assay. Engineered cell suspensions were then injected into adult Fischer 344 rat spinal cord immediately after unilateral cervical (C4) corticospinal tract (CST) transection. Transplanted animals received a total of 200,000 cells; either non-transduced OEG or OEG transduced with AdV vectors encoding NT-3 or LacZ, respectively. At 3 months after injury, lesion volumes were significantly smaller in all OEG-transplanted rats when compared with control (medium-injected) rats. Anterograde tracing of the lesioned CST projection, originating from the contralateral sensorimotor cortex, showed a significantly greater number of distal CST axons only in OEG-NT-3-transplanted rats. Behavioural analysis was performed on all rats using open field locomotion scoring, and a forelimb reaching task with Eshkol-Wachman movement notation. Analysis of behavioural tests revealed no significant differences in recovery between experimental groups, although movement analysis indicated that possible compensatory mechanisms were occurring after OEG implantation. The results demonstrate that OEG transplantation per se can promote tissue sparing after injury, but, after appropriate genetic modification, these olfactory-derived cells become far more effective in promoting long-distance maintenance/regeneration of lesioned adult CST axons.

In neonatal rats, superior colliculus (SC) ablation results in a massive and rapid increase in retinal ganglion cell (RGC) death that peaks about 24 h post-lesion (PL). Naturally occurring cell death during normal development, and RGC death after axonal injury in neonatal and adult rats, has primarily been ascribed to apoptosis. Given that normal developmental cell death is reported to involve caspase 3 activation, and blocking caspase activity in adults reduces axotomy-induced death, we examined whether blocking caspases in vivo reduces RGC death after neonatal SC lesions. Neither general nor specific caspase inhibitors increased neonatal RGC survival 6 and 24 h PL. These inhibitors were, however, effective in blocking caspases in another well-defined in vitro apoptosis model, the corpus luteum. Caspase 3 protein and mRNA levels in retinas from normal and SC-lesioned neonatal rats were assessed 3, 6 and 24 h after SC removal using immunohistochemistry, western and northern blots and quantitative real-time polymerase chain reaction. Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL) was used to independently monitor retinal cell death. The polymerase chain reaction data showed a small but insignificant increase in caspase 3 mRNA in retinas 24 h PL. Western blot analysis did not reveal a significant shift to cleaved (activated) caspase 3 protein. There was a small increase in the number of cleaved caspase 3 immunolabelled cells in the ganglion cell layer 24 h PL but this represented only a fraction of the death revealed by TUNEL. Together, these data indicate that, unlike the situation in adults, most lesion-induced RGC death in neonatal rats occurs independently of caspase activation.

We have shown previously that intraocular elevation of cAMP using the cAMP analog 8-(4-chlorophenylthio)-cAMP (CPT-cAMP) failed to promote axonal regeneration of axotomized adult retinal ganglion cells (RGCs) into peripheral nerve (PN) grafts but significantly potentiated ciliary neurotrophic factor (CNTF)-induced axonal regeneration. Using the PN graft model, we now examine the mechanisms underlying spontaneous and CNTF/CPT-cAMP-induced neuronal survival and axonal regrowth. We found that blockade of the cAMP pathway executor protein kinase A (PKA) using the cell-permeable inhibitor KT5720 did not affect spontaneous survival and axonal regeneration but essentially abolished the CNTF/CPT-cAMP-induced RGC survival and axonal regeneration. Blockade of CNTF signaling pathways such as phosphotidylinositol 3-kinase (PI3K)/akt by 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294002), mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) by 2-(2-diamino-3-methoxyphenyl-4H-1-benzopyran-4-one (PD98059), or Janus kinase (JAK)/signal transducer and activators of transcription (STAT3) by tyrphostin AG490 also blocked the CNTF/CPT-cAMP-dependent survival and regeneration effects. PKA activity assay and Western blots showed that KT5720, LY294002, and PD98059 almost completely inhibited PKA, PI3K/akt, and MAPK/ERK signal transduction, respectively, whereas AG490 substantially decreased JAK/STAT3 signal transduction. Intraocular injection of CPT-cAMP resulted in a small PKA-dependent increase in CNTF receptor α mRNA expression in the retinas, an effect that may facilitate CNTF action on survival and axonal regeneration. Surprisingly, in the absence of CNTF/CPT-cAMP, LY294002, PD98059, and AG490, but not KT5720, significantly enhanced spontaneous RGC survival, suggesting differential roles of these pathways in RGC survival under different conditions. Our data suggest that CNTF/CPT-cAMP-induced RGC survival and axonal regeneration are a result of multiple pathway actions, with PKA as an essential component, but that these pathways can function in an antagonistic manner under different conditions.

Superior colliculus (SC) ablation in neonatal rats results in a rapid increase in retinal ganglion cell (RGC) death. This injury-induced death is reduced by exogenous brain-derived neurotrophic factor or neurotrophin-4/5 (NT-4/5), but the protective effect of these molecules is transient, delaying but not preventing neuronal loss. We sought to discover why neurotrophins only temporarily reduce RGC death after target ablation, focusing on changes in neurotrophin receptor expression and possible changes in growth factor dependency. In unlesioned rats, receptor tyrosine kinase B (trkB) immunohistochemistry revealed no change in the number of trkB positive cells in the RGC layer 24 h after intraocular NT-4/5 injection. However, after SC lesions there were significantly less immunoreactive cells and, surprisingly, even fewer immunoreactive cells in NT-4/5 injected eyes. Semi-quantitative confocal analysis of immunofluorescence intensity revealed an increase in trkB staining in the RGC layer in unlesioned rats 24 h after NT-4/5 injection, whereas in SC-lesioned animals exposed to NT-4/5 there was a significant decrease in staining. To determine whether injured neonatal RGCs can switch their trophic requirements, different doses of ciliary neurotrophic factor were given intraocularly, either alone or combined with NT-4/5. We also tested an SC-derived chondroitin sulfate proteoglycan that has been reported to promote neonatal RGC survival. None of these interventions reduced lesion-induced RGC death 24 or 36 h after SC ablation. In summary, we show that developing RGCs do not shift their trophic dependence to other survival factors following injury; rather, the application of neurotrophins causes a down-regulation of the cognate trkB receptor, presumably altering the long-term responsiveness of neonatal RGCs to exogenous neurotrophins. These data highlight the difficulty in promoting long-term neuronal survival when using one-off administration of recombinant growth factors.

Brain-derived neurotrophic factor (BDNF) and neurotrophin-4/5 (NT-4/5) protein and mRNA are found in the neonatal rat retina and also in target sites such as the superficial layers of the superior colliculus. Both neurotrophins support neonatal retinal ganglion cell survival in vitro. In vivo, injections of recombinant BDNF and NT-4/5 reduce naturally occurring cell death as well as death induced by removal of the contralateral superior colliculus. In the latter case, the peak of retinal ganglion cell death occurs about 24 h postlesion. We wished to determine: whether a similar time-course of degeneration occurs after selective removal of target cells or depletion of target-derived trophic factors, and whether ganglion cell viability also depends on intraretinally derived neurotrophins. Retinal ganglion cell death was measured 24 and 48 h following injections of kainic acid or a mixture of BDNF and NT-4/5 blocking antibodies into the superior colliculus and 24 h after intraocular injection of the same antibodies. Retinotectally projecting ganglion cells were identified by retrograde labeling with the nucleophilic dye diamidino yellow. We show that collicular injections of either kainic acid or BDNF and NT-4/5 blocking antibodies significantly increased retinal ganglion cell death in the neonatal rat 24 h postinjection, death rates returning to normal by 48 h. This increase in death was greatest following collicular injections; however, death was also significantly increased 24 h following intravitreal antibody injection. Thus retinal ganglion cell survival during postnatal development is not only dependent upon trophic factors produced by central targets but may also be influenced by local intraretinal neurotrophin release.

Analysis of the effectiveness of allografts and immunosuppression in the repair of nerve defects in the adult peripheral nervous system (PNS) has a long experimental and clinical history. There is little information, however, on the use of allografts in peripheral nerve (PN) transplantation into the injured central nervous system (CNS). We assessed the ability of PN allografts (from Dark-Agouti rats) to support regeneration of adult rat retinal ganglion cell (RGC) axons in immunosuppressed host Lewis rats. PN allografts were sutured onto intraorbitally transected optic nerves. Three weeks after grafting, regenerating RGC axon numbers were determined using retrograde fluorescent labelling, and total axons within PN grafts were assessed using pan-neurofilament immunohistochemistry. In the absence of immunosuppression, PN allografts contained few axons and there were very few labelled RGC. These degenerate grafts contained many T cells and macrophages. Systemic (intraperitoneal) application of the immunosuppressants cyclosporin-A or FK506 reduced cellular infiltration into allografts and resulted in extensive axonal regrowth from surviving RGCs. The average number of RGCs regenerating axons into immunosuppressed allografts was not significantly different from that seen in PN autografts in rats sham-injected with saline. Many pan-neurofilament-positive axons, a proportion of which were myelinated, were seen in immunosuppressed allografts, particularly in proximal regions of the grafts toward the optic nerve-PN interface. This study demonstrates that PN allografts can support axonal regrowth in immunosuppressed adult hosts, and points to possible clinical use in CNS repair.

In some parts of the CNS, depletion of a particular class of neuron might induce changes in the microenvironment that influence the differentiation of newly grafted neural precursor cells. This hypothesis was tested in the retina by inducing apoptotic retinal ganglion cell (RGC) death in neonatal and adult female mice and examining whether intravitreally grafted male neural precursor cells (C17.2), a neural stem cell (NSC)-like clonal line, become incorporated into these selectively depleted retinae. In neonates, rapid RGC death was induced by removal of the contralateral superior colliculus (SC), in adults, delayed RGC death was induced by unilateral optic nerve (ON) transection. Cells were injected intravitreally 6-48 h after SC ablation (neonates) or 0-7 days after ON injury (adults). Cells were also injected into non-RGC depleted neonatal and adult retinae. At 4 or 8 weeks, transplanted cells were identified using a Y-chromosome marker and in situ hybridisation or by their expression of the lacZ reporter gene product Escherichia coli beta-galactosidase (beta-gal). No C17.2 cells were identified in axotomised adult-injected eyes undergoing delayed RGC apoptosis (n = 16). Donor cells were however stably integrated within the retina in 29% (15/55) of mice that received C17.2 cell injections 24 h after neonatal SC ablation; 6-31% of surviving cells were found in the RGC layer (GCL). These NSC-like cells were also present in intact retinae, but on average, there were fewer cells in GCL. In SC-ablated mice, most grafted cells did not express retinal-specific markers, although occasional donor cells in the GCL were immunopositive for beta-III tubulin, a protein highly expressed by, but not specific to, developing RGCs. Targeted rapid RGC depletion thus increased cell incorporation into the GCL, but grafted C17.2 cells did not appear to differentiate into an RGC phenotype.

The basic helix–loop–helix (bHLH) transcription factor stem cell leukaemia (SCL) is a ‘master regulator’ of haematopoiesis, where SCL is pivotal in cell fate determination and differentiation. SCL has also been detected in CNS, where other members of the bHLH-family have been shown to be indispensable for neuronal development; however, no detailed expression pattern of SCL has so far been described. We have generated a map of SCL expression in the embryonic and adult mouse brain based on histochemical analysis of LacZ reporter gene expression in sequential sections of brain tissue derived from SCL-LacZ knockin mice. The expression of LacZ was confirmed to reflect SCL expression by in situ hybridisation. LacZ expression was found in a range of different diencephalic, mesencephalic and metencephalic brain nuclei in adult CNS. Co-localisation of LacZ with the neuronal marker NeuN indicated expression in post-mitotic neurons in adulthood. LacZ expression by neurons was confirmed in tissue culture analysis. The nature of the pretectal, midbrain and hindbrain regions expressing LacZ suggest that SCL in adult CNS is potentially involved in processing of visual, auditory and pain related information. During embryogenesis, LacZ expression was similarly confined to thalamus, midbrain and hindbrain. LacZ staining was also evident in parts of the intermediate and marginal zone of the aqueduct and ventricular zone of the fourth ventricle at E12.5 and E14. These cells may represent progenitor stages of differentiating neural cells. Given the presence of SCL in both the developing brain and in post-mitotic neurons, it seems likely that the function of SCL in neuronal differentiation may differ from its function in maintaining the differentiated state of the mature neuron.

Rat retinal ganglion cells (RGCs) are generated between embryonic day (E) 13 and E19. Retinal axons first reach the superior colliculus at E16/16.5 but the time of arrival of axons from late-born RGCs is unknown. This study examined (i) whether there is a correlation between RGC genesis and the timing of retinotectal innervation and (ii) when axons of late-born RGCs reach the superior colliculus. Pregnant Wistar rats were injected intraperitoneally with bromodeoxyuridine (BrdU) on E16, E18 or E19. Pups from these litters received unilateral superior colliculus injections of fluorogold (FG) at ages between postnatal (P) day P0 and P6, and were perfused 1-2 days later. RGCs in 3 rats from each BrdU litter were labelled in adulthood by placing FG onto transected optic nerve. Retinas were cryosectioned and the number of FG, BrdU and double-labelled (FG+/BrdU+) RGCs quantified. In the E16 group, the proportion of FG-labelled RGCs that were BrdU+ did not vary with age, indicating that axons from these cells had reached the superior colliculus by P0/P1. In contrast, for the smaller cohorts of RGCs born on E18 or E19, the proportion of BrdU+ cells that were FG+ increased significantly after birth; axons from most RGCs born on E19 were not retrogradely FG-labelled until P4/P5. Thus there is a correlation between birthdate and innervation in rat retinotectal pathways. Furthermore, compared to the earliest born RGCs, axons from late-born RGCs take about three times longer to reach the superior colliculus. Later-arriving axons presumably encounter comparatively different growth terrains en route and eventually innervate more differentiated target structures.

Development of primary visual projections was examined in a lizard Ctenophorus ornatus by anterograde and retrograde tracing with DiI and by GAP-43 immunohistochemistry. Visual pathway development was essentially similar to that in birds and mammals and thus differed from patterns in fish or amphibians. A number of features characterised the development as mammalian-like. Three phases occurred in rapid succession after laying: outgrowth (2–3 weeks, early), exuberance (4–5 weeks, intermediate), and retraction to the adult pattern (6–8 weeks, late) at about the time of hatching and eye opening. Furthermore, ipsilateral projections developed with only a slight lag relative to the contralateral ones. The dorsally located fovea could be identified from early stages. Optic axons formed transient exuberant projections to the ipsilateral optic tectum, to the opposite optic nerve, and to nonvisual regions. The pattern resembled that formed in the long term by regenerating optic axons in C. ornatus (Dunlop et al. [2000b] J. Comp. Neurol. 416:188–200), suggesting that axons recognise molecular signals associated with the initial exuberant innervation but not those associated with subsequent refinement. J. Comp. Neurol. 453:71–84, 2002. © 2002 Wiley-Liss, Inc.

Alzheimer's disease is a progressively dementing illness characterized by the extracellular accumulation and deposition of beta-amyloid. Early onset Alzheimer's disease is linked to mutations in three genes, all of which lead to increased beta-amyloid production. Inflammatory changes and gliosis may also play a role in the disease process, but the importance of these reactive events remains unclear. We recently reported that chronic cortical gliosis in heterotopic fetal rat cortical transplants is associated with significant changes in the levels of some of the proteins implicated in the pathogenesis of Alzheimer's disease. Because rodent beta-amyloid does not form extracellular amyloid deposits, we have now extended this model of chronic cortical gliosis to transgenic mice expressing the Swedish mutant form of human amyloid precursor protein. In addition, apolipoprotein E knockout mice were used to elucidate the role of this protein in reactive gliosis. The expression of mutant and murine proteins was assayed 6 or 10 months after transplantation using immunohistochemical and western blot methods. Heterotopic transplantation of fetal cortex onto the midbrain of neonatal mice consistently resulted in reactive gliosis, independent of apolipoprotein E status. In contrast, in homotopic cortex-to-cortex grafts there was little alteration in glial reactivity, a result similar to that obtained previously in rats. By 10 months post-transplantation the level of presenilin-1 expression was lower in heterotopic grafts than in host cortex and there was increased expression of transgenic amyloid precursor protein, but only in the gliotic cortex-to-midbrain grafts. Most importantly, increased levels of beta-amyloid, and particularly its precursor, C-99, were selectively found in these heterotopic transplants. Our results show that chronic gliosis is associated with altered processing of the amyloid precursor protein in vivo and thus may initiate or exacerbate pathological changes associated with Alzheimer's disease.

Because the adult mammalian central nervous system (CNS) has only limited intrinsic capacity to regenerate connections after injury, due to factors both intrinsic and extrinsic to the mature neuron (Shen et al., 1999; Berry et al., 2008; Lingor et al., 2008; Sun and He, 2010; Moore et al., 2011), therapies are required to support the survival of injured neurons and to promote the long-distance regrowth of axons back to their original target structures. The retina and optic nerve (ON) are part of the CNS and this system is much used in experiments designed to test new ways of promoting regeneration after injury (Harvey et al., 2006; Benowitz and Yin, 2008; Berry et al., 2008; Fischer and Leibinger, 2012). Testing of therapies designed to improve retinal ganglion cell (RGC) viability also has direct clinical relevance because there is loss of these centrally projecting neurons in many ophthalmic diseases.

Implantation of olfactory ensheathing glia (OEG) is a promising strategy to augment long-distance regeneration in the injured spinal cord. In this study, implantation of OEG following unilateral hemisection of the dorsal cervical spinal cord was combined with ex vivo gene transfer techniques. We report, to our knowledge for the first time, that purified cultures of primary OEG are capable of expressing a foreign gene following adenoviral (AdV) and lentiviral (LV) vector-mediated gene transfer. OEG implants subjected to AdV vector-mediated gene transfer expressed high levels of transgenic protein in both intact and lesioned spinal cord at 7 days after implantation. However, the levels of transgene expression gradually declined between 7 and 30 days after implantation in lesioned spinal cord. Infection with LV vectors resulted in stable transduction of primary OEG cultures and transgene expression persisted for at least 4 months after implantation. Genetic engineering of OEG opens the possibility of expressing additional neurotrophic genes and create optimal 'bridging' substrates to support spinal axon regeneration. Furthermore, stable transduction of OEG allows us to reliably study the behaviour of implanted cells and to obtain better understanding of their regeneration supporting properties.

A major characteristic feature of Alzheimer's disease is the formation of compact, extracellular deposits of beta-amyloid (senile plaques). These deposits are surrounded by reactive astrocytes, microglia and dystrophic neurites. Mutations in three genes have been implicated in early-onset familial Alzheimer's disease. However, inflammatory changes and astrogliosis are also believed to play a role in Alzheimer's pathology. What is unclear is the extent to which these factors initiate or contribute to the disease progression. Previous rat studies demonstrated that heterotopic transplantation of foetal cortical tissue onto the midbrain of neonatal hosts resulted in sustained glial reactivity for many months. Similar changes were not seen in cortex-to-cortex grafts. Using this model of chronic cortical gliosis, we have now measured reactive changes in the levels of the key Alzheimer's disease proteins, namely the amyloid precursor protein, apolipoprotein E and presenilin-1. These changes were visualised immunohistochemically and were quantified by western blot analysis. We report here that chronic cortical gliosis in the rat results in a sustained increase in the levels of apolipoprotein E and total amyloid precursor protein. Reactive astrocytes in heterotopic cortical grafts were immunopositive for both of these proteins. Using a panel of amyloid precursor protein antibodies we demonstrate that chronic reactive gliosis is associated with alternative cleavage of the peptide. No significant changes in apolipoprotein E or amyloid precursor protein expression were seen in non-gliotic cortex-to-cortex transplants. Compared to host cortex, the levels of both N-terminal and C-terminal fragments of presenilin-1 were significantly lower in gliotic heterotopic grafts.The changes described here largely mirror those seen in the cerebral cortex of humans with Alzheimer's disease and are consistent with the proposal that astrogliosis may be an important factor in the pathogenesis of this disease.