Daisy Kwok-Yan Shum

Chondroitinase ABC enhances axonal regrowth through Schwann cell-seeded guidance channels after spinal cord injury

Grafting of Schwann cell‐seeded channels into hemisected adult rat thoracic spinal cords has been tested as a strategy to bridge the injured cord. Despite success in guiding axonal growth into the graft, regeneration across the distal graft–host interface into the host spinal cord was limited. We hypothesized that chondroitin sulfate (CS) glycoforms deposited at the gliotic front of the interface constitute a molecular barrier to axonal growth into the host cord. Because CS glycoforms deposited by purified astrocytes in vitro were removable by digestion with chondroitinase ABC, we attempted to achieve likewise by infusion of the enzyme to the host side of the interface. By 1 month post‐treatment, significant numbers of regenerating axons crossed an interface that was subdued in macrophage/microglia reaction and decreased in CS‐immunopositivity. The axons extended as far into the caudal cord as 5 mm, in contrast to nil in vehicle‐infused controls. Fascicular organizations of axon−Schwann cell units within the regenerated tissue cable were better‐preserved in enzyme‐treated cords than in vehicle‐infused controls. We conclude that CS glycoforms deposited during gliosis at the distal graft–host interface could be cleared by the in vivo action of chondroitinase ABC to improve prospects of axonal regeneration into the host spinal cord.

Expression of chondroitin sulfate proteoglycans in the chiasm of mouse embryos

Chondroitin sulfate (CS) proteoglycans have been implicated as molecules that are involved in axon guidance in the developing neural pathways. The spatiotemporal expression of CS was investigated in the developing retinofugal pathway in mouse embryos by using the CS‐56 antibody. Immunoreactive CS was detected in inner regions of the retina as early as embryonic day 11 (E11). Its expression in subsequent stages of development followed a centrifugal, receding gradient that appeared to correlate with the sequence of axogenesis in the retina. In the chiasm, immunoreactive CS was expressed at E12, before the arrival of retinal axons. When the retinal axons navigated in the chiasm at E13–E14, immunoreactive CS remained at a low level in the optic fiber layer of the chiasm but was observed prominently in the caudal parts of the ventral diencephalon. This pattern followed closely the array of stage‐specific‐embryonic‐antigen‐1‐positive neurons in the ventral diencephalon, with a V‐shaped configuration that bordered the posterior boundary of the retinal axons, and a rostral raphe extension that ran across the decussating axons in the chiasm. Thus, the CS epitope is implicated in patterning the course of early retinal axons and in regulating axon divergence in the chiasm. At the lateral region of the chiasm, where the retinal axons cross the midline and approach the optic tract, a CS‐immunopositive region coincided with the region in which active sorting of dorsal retinal axons from ventral retinal axons occurs. Moreover, at the threshold of the optic tract, the immunoreactive CS was restricted only to the deep part of the optic fiber layer, suggesting an inhibitory role of the CS epitope in repelling newly arrived axons to superficial regions of the optic tract during the development of chronotopic order at this part of the retinofugal pathway. J. Comp. Neurol. 417:153–163, 2000. ©2000 Wiley‐Liss, Inc.

Effect of antibiotics on sputum inflammatory contents in acute exacerbations of bronchiectasis

We studied the changes in sputum neutrophil chemotactic activity (NCA) and elastolytic activity (EA) in acute exacerbations of bronchiectasis before and after treatment with oral antibiotics. Twelve patients who chronically produced sputum were assessed in the stable state, and when they subsequently developed symptoms of acute exacerbations, prior to initiation of antibiotics, during 2 weeks of antibiotics, and at 2 and 6 weeks after stopping antibiotics. NCA was measured using modified Boydens technique with multiwell chemotaxis chamber, and EA with N-succinyl-trialanine-p-nitroanilide as elastase substrate. All 12 patients had NCA (49.3 +/- 8.69% FMLP response) and EA (50.5 +/- 17.1 mU per 100 microliters) in their sputum in the stable state. At acute exacerbation, there was significant increase in NCA (P < 0.001) and EA (P < 0.05). All responded clinically after 1 week of antibiotics, and this was associated with a decrease in NCA and EA back to the levels in stable state. A further week of antibiotics did not result in further decline of NCA or EA. Three patients had another acute exacerbation clinically between 2-6 weeks after stopping antibiotics and their NCA and EA rose again. In the other nine patients, both NCA and EA at 2 and 6 weeks post-treatment were similar to pre-exacerbation levels. Our findings suggest that short course antibiotics effectively control the upsurge in inflammatory activity in acute exacerbations, but has little effect on chronic airway inflammation.(ABSTRACT TRUNCATED AT 250 WORDS)

Localization of nerve growth factor, neurotrophin-3, and glial cell line-derived neurotrophic factor in nestin-expressing reactive astrocytes in the caudate-putamen of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-treated C57/Bl mice.

To address the hypothesis that reactive astrocytes in the basal ganglia of an animal model of Parkinsons disease serve neurotrophic roles, we studied the expression pattern of neurotrophic factors in the basal ganglia of C57/Bl mice that had been treated with 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP) to induce the degeneration of nigral dopamine neurons and parkinsonism. MPTP induced significant neuronal degeneration in the substantia nigra pars compacta as detected with Fluoro‐Jade B staining, and this was accompanied by an increase in nestin‐expressing astrocytes within the caudate‐putamen. The number of nestin‐positive reactive astrocytes in the caudate‐putamen peaked within 3–5 days following MPTP treatment and then declined progressively toward the basal level by 21 days after treatment. Immunofluorescence and confocal microscopy confirmed coexpression of nestin or Ki‐67 (cell proliferation marker) in glial fibrillary acid protein‐positive astrocytes in the caudate‐putamen. Double immunolabeling further revealed immunoreactivities for nerve growth factor (NGF), neurotrophin‐3 (NT3), and glial cell line‐derived neurotrophic factor (GDNF) in nestin‐positive reactive astrocytes. Semiquantification of data obtained from mice 5 days after MPTP injection indicated that the majority of nestin‐expressing cells expressed NGF (92%), NT3 (90%), or GDNF (86%). Our results present novel evidence of neurotrophic features among reactive astrocytes in the dopamine‐depleted striatum. These nestin‐expressing reactive astrocytes may therefore play neurotrophic roles in neural remodeling of the basal ganglia in Parkinsons disease. J. Comp. Neurol. 497:898–909, 2006.

Bone marrow-derived Schwann cells achieve fate commitment - a prerequisite for remyelination therapy

Schwann cell transplantation improves post-traumatic nerve regeneration in both PNS and CNS but sufficient numbers of immunocompatible cells are required for clinical application. Currently, Schwann cell-like cells derived from the bone marrow lack fate commitment and revert to a fibroblast-like phenotype upon withdrawal of differentiation-inducing factors. In recapitulation of embryonic events leading to Schwann cell maturation, we hypothesize that the Schwann cell-like cells acquire the switch to fate commitment through contact-dependent cues from incipient neurons of the developing dorsal root ganglia. To address this, Schwann cell-like cells derived from adult rat bone marrow were cocultured with neurons purified from embryonic dorsal root ganglia. A cell-intrinsic switch to the Schwann cell fate was achieved consistently and the cell progeny maintained expression of the markers S100 beta, p75(NTR) , GFAP, P0 and Sox 10 even without exogenous differentiation-inducing factors or neurons. In vitro formation of MBP-positive segments under myelinating conditions by the cell progeny was comparable to that by sciatic nerve-derived Schwann cells. Controls in which Schwann cell-like cells were barred from direct contact with neurons in coculture reverted to SMA/CD90-expressing myofibroblasts. We demonstrate therefore for the first time fate commitment among bone marrow-derived Schwann cells. The therapeutic potential of these cells may be tested in future transplantation studies. (206 words).

Fos expression in otolith-related brainstem neurons of postnatal rats following off-vertical axis rotation

To determine the critical time of responsiveness of developing otolith organ‐related brainstem neurons and their distribution, Fos protein expression in response to off‐vertical axis rotations (OVAR) was mapped in conscious Sprague Dawley rats from P5 to adulthood. OVAR was used to activate sequentially all utricular hair cells per 360° revolution. We detected the coding of horizontal head positions in otolith organ‐related neurons within the vestibular nucleus as early as P7. In the vestibular nuclear complex and its subgroups, the density of Fos‐immunoreactive (Fos‐ir) neurons increased steadily with age and reached the adult level by P21. In both labyrinthectomized rats subjected to OVAR and normal rats kept stationary, labeled neurons were found sporadically in the aforementioned brain regions in each age group, confirming that Fos labeling observed in neurons of normal experimental rats subjected to OVAR was due to otolith organ stimulation. Whereas OVAR‐induced Fos‐ir neurons were also first observed in vestibular‐related brain areas, such as the prepositus hypoglossal nucleus, gigantocellular reticular nucleus, and locus coeruleus, of normal experimental rats at P7, those in the inferior olive were observed only from P14 onward. This indicates the unique maturation time of inferior olivary neurons in gravity‐related spatial coding. In general, age‐dependent increase in OVAR‐induced Fos‐ir neurons was observed in brain areas that received otolith inputs. The locus coeruleus was exceptional in that prominent OVAR‐induced Fos‐ir neuronal number did not change with maturation, and this was well above the low but significant number of Fos‐ir neurons in control preparations. Taken together, our results suggest that neuronal subpopulations within the developing network of the horizontal otolith system provide an anatomical basis for the postnatal development of otolith organ‐related sensorimotor functions. J. Comp. Neurol. 470:282–296, 2004.

Developmental expression of NMDA and AMPA receptor subunits in vestibular nuclear neurons that encode gravity-related horizontal orientations.

We examined the expression profile of subunits of ionotropic glutamate receptors [N‐methyl‐D‐aspartate (NMDA) and α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazole‐proprionate (AMPA)] during postnatal development of connectivity in the rat vestibular nucleus. Vestibular nuclear neurons were functionally activated by constant velocity off‐vertical axis rotation, a strategy to stimulate otolith organs in the inner ear. These neurons indicated Fos expression as a result. By immunodetection for Fos, otolith‐related neurons that expressed NMDA/AMPA receptor subunits were identified as early as P7, and these neurons were found to increase progressively up to adulthood. Although there was developmental invariance in the percentage of Fos‐immunoreactive neurons expressing the NR1, NR2A, GluR1, or GluR2/3 subunits, those expressing the NR2B subunit decreased from P14 onward, and those expressing the GluR4 subunit decreased in adults. These double‐immunohistochemical data were corroborated by combined immuno‐/hybridization histochemical data obtained from Fos‐immunoreactive neurons expressing NR2B mRNA or GluR4 mRNA. The staining of both NR2B and GluR4 in the cytoplasm of these neurons decreased upon maturation. The percentage of Fos‐immunoreactive neurons expressing the other ionotropic glutamate receptor subunits (viz. NR1, NR2A, GluR1, and GluR2/3) remained relatively constant throughout postnatal maturation. Triple immunofluorescence further demonstrated coexpression of NR1 and NR2 subunits in Fos‐immunoreactive neurons. Coexpression of NR1 subunit with each of the GluR subunits was also observed among the Fos‐immunoreactive neurons. Taken together, the different expression profiles of ionotropic glutamate receptor subunits constitute the histological basis for glutamatergic neurotransmission in the maturation of central vestibular connectivity for the coding of gravity‐related horizontal head movements. J. Comp. Neurol. 508:343–364, 2008.

Corticofugal modulation of acoustically induced Fos expression in the rat auditory pathway

To investigate the corticofugal modulation of acoustic information ascending through the auditory pathway of the rat, immunohistochemical techniques were used to study the functional expression of Fos protein in neurons. With auditory stimulation at different frequencies, Fos expression in the medial geniculate body (MGB), inferior colliculus (IC), superior olivary complex, and cochlear nucleus was examined, and the extent of Fos expression on the two sides was compared. Strikingly, we found densely Fos‐labeled neurons in all divisions of the MGB after both presentation of an auditory stimulus and administration of a γ‐aminobutyric acid type A (GABAA) antagonist (bicuculline methobromide; BIM) to the auditory cortex. The location of Fos‐labeled neurons in the ventral division (MGv) after acoustic stimulation at different frequencies was in agreement with the known tonotopic organization. That no Fos‐labeled neurons were found in the MGv with acoustic stimuli alone suggests that the transmission of ascending thalamocortical information is critically governed by corticofugal modulation. The dorsal (DCIC) and external cortices (ECIC) of the IC ipsilateral to the BIM‐injected cortex showed a significantly higher number of Fos‐labeled neurons than the contralateral IC. However, no difference in the number of Fos‐labeled neurons was found between the central nucleus of the IC on either side, indicating that direct corticofugal modulation occurs only in the ECIC and DCIC. Further investigations are needed to assess the functional implications of the morphological differences observed between the descending corticofugal projections to the thalamus and the IC. J. Comp. Neurol. 501:509–525, 2007.

Upregulation of chondroitin 6-sulphotransferase-1 facilitates Schwann cell migration during axonal growth

Cell migration is central to development and post-traumatic regeneration. The differential increase in 6-sulphated chondroitins during axonal growth in both crushed sciatic nerves and brain development suggests that chondroitin 6-sulphotransferase-1 (C6ST-1) is a key enzyme that mediates cell migration in the process. We have cloned the cDNA of the C6ST-1 gene (C6st1) (GenBank accession number AF178689) from crushed sciatic nerves of adult rats and produced ribonucleotide probes accordingly to track signs of 6-sulphated chondroitins at the site of injury. We found C6st1 mRNA expression in Schwann cells emigrating from explants of both sciatic nerve segments and embryonic dorsal root ganglia. Immunocytochemistry indicated pericellular 6-sulphated chondroitin products around C6ST-1-expressing frontier cells. Motility analysis of frontier cells in cultures subjected to staged treatment with chondroitinase ABC indicated that freshly produced 6-sulphated chondroitin moieties facilitated Schwann cell motility, unlike restrictions resulting from proteoglycan interaction with matrix components. Sciatic nerve crush provided further evidence of in vivo upregulation of the C6ST-1 gene in mobile Schwann cells that guided axonal regrowth 1-14 days post crush; downregulation then accompanied declining mobility of Schwann cells as they engaged in the myelination of re-growing axons. These findings are the first to identify upregulated C6st1 gene expression correlating with the motility of Schwann cells that guide growing axons through both developmental and injured environments.

Shed syndecan-1 restricts neutrophil elastase from α1- antitrypsin in neutrophilic airway inflammation

Persistent proteolytic imbalance in chronic inflammatory diseases has been ascribed to neutrophil elastase (NE)/antielastase imbalance in wound fluids. In sputum sols of patients with bronchiectasis, we found unopposed NE activity, despite overwhelming excess of the physiological antielastase, alpha(1)-antitrypsin (alpha(1)-AT). Western blot analysis found NE in a supramolecular complex with shed ectodomains of syndecan (Syn)-1 in sputum sol samples and, as such, inhibition of NE activity was incomplete, even with addition of exogenous alpha(1)-AT. To confirm that NE binding to heparan sulfate (HS) components of Syn-1 limits the antielastase effect, recombinant human Syn-1 was recovered from stable Syn-1 transfectants of a human B-lymphoid cell line (ARH-77). Western ligand blot confirmed that NE bound to HS moieties and alpha(1)-AT to the core protein of the recombinant product. Inhibition of NE activity by standard additions of alpha(1)-AT was incomplete unless Syn-1 had been deglycanated by heparitinase treatment. Surface plasmon resonance analysis revealed that NE binding to HS (equilibrium dissociation constant, approximately 14 nM) could be outcompeted by heparin variants. We conclude that the HS moiety of shed Syn-1 binds and restricts NE from inhibition by alpha(1)-AT.