Pseudomonas aeruginosa Infection Modulates the Immune Response and Increases Mice Resistance to Cryptococcus gattii.

Cryptococcosis is an invasive mycosis caused by Cryptococcus spp. that affects the lungs and the central nervous system (CNS). Due to the severity of the disease, it may occur concomitantly with other pathogens, as a coinfection. Pseudomonas aeruginosa (Pa), an opportunistic pathogen, can also cause pneumonia. In this work, we studied the interaction of C. gattii (Cg) and Pa, both in vitro and in vivo. Pa reduced growth of Cg by the secretion of inhibitory molecules in vitro. Macrophages previously stimulated with Pa presented increased fungicidal activity. In vivo, previous Pa infection reduced morbidity and delayed the lethality due to cryptococcosis. This phenotype was correlated with the decreased fungal burden in the lungs and brain, showing a delay of Cg translocation to the CNS. Also, there was increased production of IL-1ß, CXCL-1, and IL-10, together with the influx of iNOS-positive macrophages and neutrophils to the lungs. Altogether, Pa turned the lung into a hostile environment to the growth of a secondary pathogen, making it difficult for the fungus to translocate to the CNS. Further, iNOS inhibition reverted the Pa protective phenotype, suggesting its important role in the coinfection. Altogether, the primary Pa infection leads to balanced pro-inflammatory and anti-inflammatory responses during Cg infection. This response provided better control of cryptococcosis and was decisive for the mild evolution of the disease and prolonged survival of coinfected mice in a mechanism dependent on iNOS.

17-ß-Estradiol increases macrophage activity through activation of the G-protein-coupled estrogen receptor and improves the response of female mice to Cryptococcus gattii.

Cryptococcus gattii (Cg) is one of the agents of cryptococcosis, a severe systemic mycosis with a higher prevalence in men than women, but the influence of the female sex hormone, 17-ß-estradiol (E2), on cryptococcosis remains unclear. Our study shows that female mice presented delayed mortality, increased neutrophil recruitment in bronchoalveolar lavage fluid, and reduced fungal load after 24 hr of infection compared to male and ovariectomised female mice (OVX). E2 replacement restored OVX female survival. Female macrophages have more efficient fungicidal activity, which was increased by E2 and reversed by the antagonist of G-protein-coupled oestrogen receptor (GPER), which negatively modulates PI3K activation. Furthermore, E2 induces a reduction in Cg cell diameter, cell charge, and antioxidant peroxidase activity. In conclusion, female mice present improved control of Cg infection, and GPER is important for E2 modulation of the female response.

Spatially resolved common-path high-order harmonic interferometry.

Spatially resolved interference is observed between high-order harmonics generated in two longitudinally separated gas targets. High-contrast modulations in the intensity of each harmonic order up to the cutoff are observed on-axis in the far field of the source as the separation between the gas targets is increased. For low-order harmonics, additional off-axis modulations are observed, which are attributed to the interference between the contributions from the long quantum trajectories from each gas target. The inherent synchronization of this setup offers the prospect for high-stability metrology of quantum states with ultrafast temporal resolutions.

Evaluation of the potential antimicrobial resistance transfer from a multi-drug resistant Escherichia coli to Salmonella in dairy calves.

Previous research conducted in our laboratory found a significant prevalence of multi-drug resistant (MDR) Salmonella and MDR Escherichia coli (MDR EC) in dairy calves and suggests that the MDR EC population may be an important reservoir for resistance elements that could potentially transfer to Salmonella. Therefore, the objective of the current research was to determine if resistance transfers from MDR EC to susceptible strains of inoculated Salmonella. The experiment utilized Holstein calves (approximately 3 weeks old) naturally colonized with MDR EC and fecal culture negative for Salmonella. Fecal samples were collected for culture of Salmonella and MDR EC throughout the experiment following experimental inoculation with the susceptible Salmonella strains. Results initially suggested that resistance did transfer from the MDR E. coli to the inoculated strains of Salmonella, with these stains demonstrating resistance to multiple antibiotics following in vivo exposure to MDR EC. However, serogrouping and serotyping results from a portion of the Salmonella isolates recovered from the calves post-challenge, identified two new strains of Salmonella; therefore transfer of resistance was not demonstrated under these experimental conditions.

The effect of dietary protein level on performance characteristics of coccidiosis vaccinated and nonvaccinated broilers following mixed-species Eimeria challenge.

A series of experiments were conducted to investigate the effect of starter diet protein levels on the performance of broilers vaccinated with a commercially available live oocyst coccidiosis vaccine before subsequent challenge with a mixed-species Eimeria challenge. Data indicated that an increasing protein concentration in the starter diet improved broiler performance during coccidiosis vaccination. Prechallenge performance data indicated that vaccination could decrease BW and increase feed conversion ratio. The time period most important for the observed effects appeared to be between 13 and 17 d of age. This reduction in performance parameters of vaccinated broilers compared with nonvaccinated broilers was eliminated by the conclusion of the experiments (27 d) in the diet groups with higher protein. Vaccination was effective at generating protective immunity against Eimeria challenge, as evidenced by increased (P < 0.05) BW gain, improved feed conversion, reduced postchallenge mortality, and reduced lesion development in vaccinated broilers compared with nonvaccinated broilers. These observations support numerous other reports that confirm live oocyst vaccination can be used effectively as a preventive against avian coccidiosis in commercially reared broilers. More important, these findings suggest that reduced protein concentration of starter diets can lead to significant losses in broiler performance when using a vaccination program to prevent coccidiosis.

Molecular analysis of Salmonella serotypes at different stages of commercial turkey processing.

Salmonella isolates were collected from 2 commercial turkey processing plants (A and B) located in different US geographical locations. Isolates recovered at different stages of processing were subjected to 2 genotype techniques [PAGE and denatured gradient gel electrophoresis (DGGE)] to determine their usefulness for Salmonella serotyping. Primers used for PCR amplification were to a highly conserved spacer region located between the 16S and 23S rDNA genes. Sampling sites at plant A were 1) postscald, 2) pre-inside-outside bird wash, 3) post-IOBW, and 4) postchill with 30, 44, 36, and 12 Salmonella isolates recovered, respectively. Plant B had an additional site and these locations were 1) prescald, 2) postscald, 3) pre-inside-outside bird wash, 4) post-IOBW, and 5) postchill with 16, 54, 24, 35, and 24 Salmonella isolates recovered, respectively. In plant A, 4 different Salmonella serotypes were identified: Derby, Hadar, Montevideo, and Senftenberg. In plant B, 10 serotypes were identified: Agona, Anatum, Brandenburg, Derby, Hadar, Meleagridis, Montevideo, Reading, Senftenberg, and Typhimurium. Salmonella Derby was predominant in plant A (83%), whereas Salmonella Typhimurium was the most common serotype recovered in plant B (39%). Genotype analyses of the Salmonella serotypes were expressed in dendrograms with comparisons interpreted as percentage similarity coefficients. Both PAGE and DGGE were able to distinguish serotype band patterns. However, DGGE was more discriminating than PAGE. Isolates of the same serotypes were grouped together on the dendrogram of band patterns generated by DGGE. In contrast, PAGE failed to group all like serotypes together on the corresponding dendrogram. The results of the study suggest that genotyping techniques can be very useful in discriminating Salmonella serotypes collected from the processing plant environment of commercial poultry production. These molecular techniques may offer more cost-effective means to identify Salmonella serotypes from large numbers of isolates and with more immediate results than those currently achieved with conventional typing techniques.

Evaluation of repetitive extragenic palindromic-polymerase chain reaction and denatured gradient gel electrophoresis in identifying Salmonella serotypes isolated from processed turkeys.

The current study was conducted to determine the usefulness of 2 molecular techniques, automated repetitive extragenic palindromic-PCR (REP-PCR) and denaturing gradient gel electrophoresis (DGGE), to identify Salmonella serotypes of poultry origin. Salmonella continues to be a foodborne pathogen of principal concern in the United States. The interspersed conserved repetitive sequence of the bacterial genome and the 16-23S rDNA intergenic spacer region were amplified for REP-PCR and DGGE, respectively. Fifty-four Salmonella isolates from 2 turkey processing plants (A and B) were used for this comparison. Serotypes consisted of Brandenburg, Derby, Hadar, and Typhimurium, with n=6, 21, 12, and 15, respectively. The REP-PCR was fully automated, whereas DGGE was run on an acrylamide gel and the image was captured digitally. Both dendrograms were created using the unweighted pair group method with arithmetic average. There were more variations in percentage similarity in DGGE when compared with REP-PCR. The banding patterns were more distinct and uniform in the REP-PCR group than with DGGE. The results from the REP-PCR were generated within 1 h, whereas the DGGE required approximately 1 d to run. These data suggest that DGGE and REP-PCR are useful tools for identifying Salmonella serotypes isolated from poultry production or processing environments. In addition, REP-PCR is more rapid, may have a higher discriminatory power, but may be less cost-effective than DGGE. However, more research may be needed to validate this argument. Both DGGE and REP-PCR displayed high sensitivity in discriminating among Salmonella serotypes and either method could be considered as an alternative to more expensive and time-consuming conventional antibody-based serotyping methodologies.

Efficient gene delivery to the adult and fetal CNS using pseudotyped non-integrating lentiviral vectors.

Non-integrating lentiviral vectors show considerable promise for gene therapy applications as they persist as long-term episomes in non-dividing cells and diminish risks of insertional mutagenesis. In this study, non-integrating lentiviral vectors were evaluated for their use in the adult and fetal central nervous system of rodents. Vectors differentially pseudotyped with vesicular stomatitis virus, rabies and baculoviral envelope proteins allowed targeting of varied cell populations. Efficient gene delivery to discrete areas of the brain and spinal cord was observed following stereotactic administration. Furthermore, after direct in utero administration (E14), sustained and strong expression was observed 4 months into adulthood. Quantification of transduction and viral copy number was comparable when using non-integrating lentivirus and conventional integrating vector. These data support the use of non-integrating lentiviral vectors as an effective alternative to their integrating counterparts in gene therapy applications, and highlight their potential for treatment of inherited and acquired neurological disorders.

Retrograde viral transduction of cortical pyramidal neurons from the spinal cord.

PURPOSE: The primary motor pathway, the corticospinal tract, is a major target for spinal cord regeneration studies. One way of improving the regeneration of corticospinal axons is to introduce regeneration-associated genes into cortical motor neurons using viral vector delivery. METHODS: We used an engineered Herpes Simplex virus (HSV1) with the EF1alpha promoter encoding either LacZ or GFP to transduce cortical neurons through retrograde transport following the injection of vector into adult rat striatum or spinal cord. After three-days to one-month post-injection, sections of brain and spinal cord were viewed with fluorescence microscopy or processed for LacZ histochemistry. RESULTS: Many layer V motor cortical neurons were transduced following striatal injections. These were not corticospinal neurons as they were not fluorogold-labelled following tracer injection into spinal cord. Corticospinal neurons in both hemispheres were, however, transduced following direct vector injections into the dorsal column of spinal cord, yielding 250-400 transduced corticospinal neurons per animal. No non-pyramidal neurons or thalamic neurons were transduced by spinal injections. CONCLUSIONS: Therefore, this HSV1.EF1alpha vector is highly effective for the transduction of corticospinal neurons without direct injection into the brain and could be used to introduce regeneration-relevant genes into these neurons with the aim of regenerating the corticospinal tract.

Axonal guidance molecules and the failure of axonal regeneration in the adult mammalian spinal cord.

A wide variety of molecules are involved as attractive or repulsive guidance cues in the developing nervous system. Some of these molecules are also expressed in the CNS of adult mammals where, following injury, they may repel regenerating axons, inhibit axonal regrowth, or control the behaviour of other cells important for the development of the meningeal and glial scars or the immune response to injury. Ephrins, semaphorins, Slits, Netrins, bone morphogenetic proteins (BMPs) and Wnts are among the most likely molecules to be involved in limiting axonal regeneration in the injured spinal cord. The receptors for these molecules are not universally expressed by neurons but there is evidence that ephrins and semaphorins limit regeneration of particular classes of axon into spinal cord lesion sites. It is likely that other repulsive guidance cues will also differentially affect the regeneration of specific tracts within the spinal cord. In addition to direct effects on axonal regeneration, many axonal guidance molecules have effects on glial, meningeal or immune system cells which also modulate the responses of CNS tissue to injury.

Effects of lipopolysaccharide-induced inflammation on expression of growth-associated genes by corticospinal neurons.

BACKGROUND: Inflammation around cell bodies of primary sensory neurons and retinal ganglion cells enhances expression of neuronal growth-associated genes and stimulates axonal regeneration. We have asked if inflammation would have similar effects on corticospinal neurons, which normally show little response to spinal cord injury. Lipopolysaccharide (LPS) was applied onto the pial surface of the motor cortex of adult rats with or without concomitant injury of the corticospinal tract at C4. Inflammation around corticospinal tract cell bodies in the motor cortex was assessed by immunohistochemistry for OX42 (a microglia and macrophage marker). Expression of growth-associated genes c-jun, ATF3, SCG10 and GAP-43 was investigated by immunohistochemistry or in situ hybridisation. RESULTS: Application of LPS induced a gradient of inflammation through the full depth of the motor cortex and promoted c-Jun and SCG10 expression for up to 2 weeks, and GAP-43 upregulation for 3 days by many corticospinal neurons, but had very limited effects on neuronal ATF3 expression. However, many glial cells in the subcortical white matter upregulated ATF3. LPS did not promote sprouting of anterogradely labelled corticospinal axons, which did not grow into or beyond a cervical lesion site. CONCLUSION: Inflammation produced by topical application of LPS promoted increased expression of some growth-associated genes in the cell bodies of corticospinal neurons, but was insufficient to promote regeneration of the corticospinal tract.

Upregulation of activating transcription factor 3 (ATF3) by intrinsic CNS neurons regenerating axons into peripheral nerve grafts.

The expression of the transcription factor ATF3 in the brain was examined by immunohistochemistry during axonal regeneration induced by the implantation of pieces of peripheral nerve into the thalamus of adult rats. After 3 days, ATF3 immunoreactivity was present in many cells within approximately 500 mum of the graft. In addition, ATF3-positive cell nuclei were found in the thalamic reticular nucleus (TRN) and medial geniculate nuclear complex (MGN), from which most regenerating axons originate. CNS cells with ATF3-positive nuclei were predominantly neurons and did not show signs of apoptosis. The number of ATF3-positive cells had declined by 7 days and further by 1 month after grafting when most ATF3-positive cells were found in the TRN and MGN. 14 days or more after grafting, some ATF3-positive nuclei were distorted and may have been apoptotic. In some experiments of 1 month duration, neurons which had regenerated axons to the distal ends of grafts were retrogradely labeled with DiAsp. ATF3-positive neurons in these animals were located in regions of the TRN and MGN containing retrogradely labeled neurons and the great majority were also labeled with DiAsp. SCG10 and c-Jun were found in neurons in the same regions as retrogradely labeled and ATF3-positive cells. Thus, ATF3 is transiently upregulated by injured CNS neurons, but prolonged expression is part of the pattern of gene expression associated with axonal regeneration. The co-expression of ATF3 with c-jun suggests that interactions between these transcription factors may be important for controlling the program of gene expression necessary for regeneration.

ATF3 upregulation in glia during Wallerian degeneration: differential expression in peripheral nerves and CNS white matter.

BACKGROUND: Many changes in gene expression occur in distal stumps of injured nerves but the transcriptional control of these events is poorly understood. We have examined the expression of the transcription factors ATF3 and c-Jun by non-neuronal cells during Wallerian degeneration following injury to sciatic nerves, dorsal roots and optic nerves of rats and mice, using immunohistochemistry and in situ hybridization. RESULTS: Following sciatic nerve injury--transection or transection and reanastomosis--ATF3 was strongly upregulated by endoneurial, but not perineurial cells, of the distal stumps of the nerves by 1 day post operation (dpo) and remained strongly expressed in the endoneurium at 30 dpo when axonal regeneration was prevented. Most ATF3+ cells were immunoreactive for the Schwann cell marker, S100. When the nerve was transected and reanastomosed, allowing regeneration of axons, most ATF3 expression had been downregulated by 30 dpo. ATF3 expression was weaker in the proximal stumps of the injured nerves than in the distal stumps and present in fewer cells at all times after injury. ATF3 was upregulated by endoneurial cells in the distal stumps of injured neonatal rat sciatic nerves, but more weakly than in adult animals. ATF3 expression in transected sciatic nerves of mice was similar to that in rats. Following dorsal root injury in adult rats, ATF3 was upregulated in the part of the root between the lesion and the spinal cord (containing Schwann cells), beginning at 1 dpo, but not in the dorsal root entry zone or in the degenerating dorsal column of the spinal cord. Following optic nerve crush in adult rats, ATF3 was found in some cells at the injury site and small numbers of cells within the optic nerve displayed weak immunoreactivity. The pattern of expression of c-Jun in all types of nerve injury was similar to that of ATF3. CONCLUSION: These findings raise the possibility that ATF3/c-Jun heterodimers may play a role in regulating changes in gene expression necessary for preparing the distal segments of injured peripheral nerves for axonal regeneration. The absence of the ATF3 and c-Jun from CNS glia during Wallerian degeneration may limit their ability to support regeneration.

NG2 proteoglycan expression in the peripheral nervous system: upregulation following injury and comparison with CNS lesions.

The chondroitin sulphate proteoglycan NG2 blocks neurite outgrowth in vitro and thus may be able to inhibit axonal regeneration in the CNS. We have used immunohistochemistry to compare the expression of NG2 in the PNS, where axons regenerate, and the spinal cord, where regeneration fails. NG2 is expressed by satellite cells in dorsal root ganglia (DRG) and in the perineurium and endoneurium of intact sciatic nerves of adult rats. Endoneurial NG2-positive cells were S100-negative. Injury to dorsal roots, ventral rami or sciatic nerves had no effect on NG2 expression in DRG but sciatic nerve section or crush caused an upregulation of NG2 in the damaged nerve. Strongly NG2-positive cells in damaged nerves were S100-negative. The proximal stump of severed nerves was capped by dense NG2, which surrounded bundles of regenerating axons. The distal stump, into which axons regenerated, also contained many NG2-positive/S100-negative cells. Immunoelectron microscopy revealed that most NG2-positive cells in distal stumps had perineurial or fibroblast-like morphologies, with NG2 being concentrated at the poles of the cells in regions exhibiting microvillus-like protrusions or caveolae. Compression and partial transection injuries to the spinal cord also caused an upregulation of NG2, and NG2-positive cells and processes invaded the lesion sites. Transganglionically labelled ascending dorsal column fibres, stimulated to sprout by a conditioning sciatic nerve injury, ended in the borders of lesions among many NG2-positive processes. Thus, NG2 upregulation is a feature of the response to injury in peripheral nerves and in the spinal cord, but it does not appear to limit regeneration in the sciatic nerve.

Nogo-A expression in the intact and injured nervous system.

The expression of Nogo-A mRNA and protein in the nervous system of adult rats and cultured neurons was studied by in situ hybridisation and immunohistochemistry. Nogo-A mRNA was expressed by many cells in unoperated animals, including spinal motor, DRG, and sympathetic neurons, retinal ganglion cells, and neocortical, hippocampal, and Purkinje neurons. Nogo-A protein was strongly expressed by presumptive oligodendrocytes, but not by NG2+glia and was abundant in motor, DRG, and sympathetic neurons, retinal ganglion cells, and many Purkinje cells, but was difficult to detect in dentate gyrus neurons and some neocortical neurons. Cultured fetal mouse neocortical neurons and adult rat DRG neurons strongly expressed Nogo-A in their perikarya, growth cones, and axonal varicosities. All axons in the intact sciatic nerve contained Nogo-A and many but not all regenerating axons were strongly Nogo-A immunopositive after sciatic nerve transection. Ectopic muscle fibres that developed among the regenerating axons were also Nogo-A immunopositive. Following injury to the spinal cord, Nogo-A mRNA was upregulated around the lesion and Nogo-A protein was strongly expressed in injured dorsal column fibres and their sprouts which entered the lesion site. Following optic nerve crush, Nogo-A accumulated in the proximal and distal stumps bordering the lesions.

Corticospinal neurons up-regulate a range of growth-associated genes following intracortical, but not spinal, axotomy.

The failure of some CNS neurons to up-regulate growth-associated genes following axotomy may contribute to their failure to regenerate axons. We have studied gene expression in rat corticospinal neurons following either proximal (intracortical) or distal (spinal) axotomy. Corticospinal neurons were retrogradely labelled with cholera toxin subunit B prior to intracortical lesions or concomitantly with spinal lesions. Alternate sections of forebrain were immunoreacted for cholera toxin subunit B or processed for mRNA in situ hybridization for ATF3, c-jun, GAP-43, CAP-23, SCG10, L1, CHL1 or krox-24, each of which has been associated with axotomy or axon regeneration in other neurons. Seven days after intracortical axotomy, ATF3, c-jun, GAP-43, SCG10, L1 and CHL1, but not CAP-23 or krox-24, were up-regulated by layer V pyramidal neurons, including identified corticospinal neurons. The maximum distance between the lesion and the neuronal cell bodies that up-regulated genes varied between 300 and 500 microm. However, distal axotomy failed to elicit changes in gene expression in corticospinal neurons. No change in expression of any molecule was seen in the neocortex 1 or 7 days after corticospinal axotomy in the cervical spinal cord. The expression of GAP-43, CAP-23, L1, CHL1 and SCG10 was confirmed to be unaltered after this type of injury in identified retrogradely labelled corticospinal neurons. Thus, while corticospinal neuronal cell bodies fail to respond to spinal axotomy, these cells behave like regeneration-competent neurons, up-regulating a wide range of growth-associated molecules if axotomized within the cerebral cortex.

FKBP12 mRNA expression is upregulated by intrinsic CNS neurons regenerating axons into peripheral nerve grafts in the brain.

We have examined the expression of the immunophilin FKBP12 in adult rat intrinsic CNS neurons stimulated to regenerate axons by the implantation of segments of autologous tibial nerve into the thalamus or cerebellum. After survival times of 3 days to 6 weeks, the brains were fresh-frozen. In some animals the regenerating neurons were retrogradely labelled with cholera toxin subunit B 1 day before they were killed. Sections through the thalamus or cerebellum were used for in situ hybridization with digoxygenin-labelled riboprobes for FKBP12 or immunohistochemistry to detect cholera toxin subunit B-labelled neurons. FKBP12 was constitutively expressed by many neurons, and was very strongly expressed in the hippocampus and by Purkinje cells. Regenerating neurons were found in the thalamic reticular nucleus and deep cerebellar nuclei of animals that received living grafts. Neurons in these nuclei upregulated FKBP12 mRNA; such neurons were most numerous at 3 days post grafting but were most strongly labelled at 2 weeks post grafting. Regenerating neurons identified by retrograde labelling were found to have upregulated FKBP12 mRNA. No upregulation was seen in neurons in animals that received freeze-killed grafts, which do not support axonal regeneration. We conclude that FKBP12 is a regeneration-associated gene in intrinsic CNS neurons.

Expression of regeneration-related molecules in injured and regenerating striatal and nigral neurons.

Peripheral nerve grafts in the neostriatum promote axonal regeneration from restricted classes of CNS neuron, principally cells in the substantia nigra pars compacta (SNpc) and striatal cholinergic interneurons. We have examined the molecular responses of CNS neurons induced to regenerate axons by tibial nerve grafting to the neostriatum of adult rats. Brain sections were probed for mRNAs for the transcription factor c-jun, and the cell recognition molecule CHL1, or immunoreacted for TrkA or p75, 1 day to 29 weeks after grafting (dpo; wpo). In unoperated rats, scattered neurons throughout the neostriatum showed weak signals for CHL1 mRNA and slightly stronger signals for c-jun mRNA. Cells of similar appearance strongly expressed TrkA but possessed little p75. By 1 dpo, many neostriatal neurons of various sizes and GFAP + glial cells near the host/graft interface had upregulated CHL1 mRNA, c-jun mRNA and p75. Most of the larger (20-25 microm diameter) CHL1 mRNA+ cells were also TrkA+, indicating that they were NGF-sensitive cholinergic interneurons. From two weeks postgrafting, high levels of CHL1 and c-jun mRNAs and p75 in the neostriatum were confined to a few presumptive cholinergic interneurons; p75+ cells were also TrkA+ and were larger than TrkA+ neurons on the contralateral side. Retrograde labelling showed that most p75+ and some TrkA+ neurons regenerated axons through the graft. Neurons in the SNpc showed a moderate to strong signal for CHL1 mRNA, weaker signal for c-jun mRNA, and no p75 or TrkA. Some SNpc cells upregulated c-jun mRNA after graft implantation, although they did not upregulate CHL1 mRNA, p75 or TrkA. Since neostriatal neurons which regenerate axons into grafts express receptors for NGF, and grafts mimic the effects of NGF treatment on these cells, sensitivity to graft-derived NGF may be a determinant of their high regenerative capacity. The finding that c-jun and CHL1 are consistently expressed by CNS neurons induced to regenerate their axons strongly supports the idea that these molecules are directly involved in axonal regeneration.

Nogo receptor mRNA expression in intact and regenerating CNS neurons.

The expression of mRNA for Nogo-66 receptor (NgR) in unoperated adult rats and mice, and rats with nerve grafts placed in the thalamus and cerebellum to stimulate axonal regeneration, was investigated by in situ hybridization. NgR was strongly expressed in neurons of the neocortex, hippocampal formation, and amygdaloid nuclei and dorsal thalamus and moderately expressed in the red nucleus and vestibular nuclei. NgR mRNA was expressed in cerebellar deep nuclei and more strongly by granule cells than by Purkinje cells. Large regions of the forebrain, including the striatum, thalamic reticular nucleus, hypothalamus, and basal forebrain showed little or no NgR expression. NgR was weakly expressed in spinal neurons and some primary sensory neurons. Nerve implantation into the brain did not affect NgR expression. Some regeneration-competent neurons expressed NgR but others did not. Thus NgR expression was not correlated with the ability of neurons to regenerate axons into nerve grafts although Nogo-66 was strongly upregulated by some cells in the distal stumps of injured sciatic nerves. Nogo-66 transcripts were strongly expressed by many classes of CNS neurons and less strongly in white matter.

Transcriptional upregulation of SCG10 and CAP-23 is correlated with regeneration of the axons of peripheral and central neurons in vivo.

We have compared SCG10 and CAP-23 expression with that of GAP-43 during axonal regeneration in the peripheral and central nervous systems (PNS, CNS) of adult rats. SCG10, CAP-23, and GAP-43 mRNAs were strongly upregulated by motor and dorsal root ganglion (DRG) neurons following sciatic nerve crush, but not after dorsal rhizotomy. When the sciatic nerve was cut and ligated to prevent reinnervation of targets, expression of all three mRNAs was prolonged. Neurons in the thalamic reticular nucleus and deep cerebellar nuclei transiently upregulated these mRNAs after axotomy, and showed prolonged upregulation of all three molecules when regenerating axons into peripheral nerve grafts inserted into the thalamus of cerebellum. Neurons in the dorsal thalamus and cerebellar cortex showed poor regenerative capacity and most did not upregulate any of these mRNAs. Thus, in both PNS and CNS neurons, the transcription of SCG10, CAP-23, and GAP-43 mRNAs is coregulated following axotomy and during regeneration. Signals from living peripheral nerve appear to maintain expression of all three mRNAs in regenerating neurons, and in PNS neurons downregulation correlates with target reinnervation. Thus, SCG10 and CAP-23, as well as GAP-43, are likely to be important neuronal determinants of regenerative ability.