Prevention against diffuse spinal cord astrocytoma: can the Notch pathway be a novel treatment target?

This study was designed to investigate whether the Notch pathway is involved in the development of diffuse spinal cord astrocytomas. BALB/c nude mice received injections of CD133+ and CD133- cell suspensions prepared using human recurrent diffuse spinal cord astrocytoma tissue through administration into the right parietal lobe. After 7–11 weeks, magnetic resonance imaging was performed weekly. Xenografts were observed on the surfaces of the brains of mice receiving the CD133+ cell suspension, and Notch-immunopositive expression was observed in the xenografts. By contrast, no xenografts appeared in the identical position on the surfaces of the brains of mice receiving the CD133- cell suspension, and Notch-immunopositive expression was hardly detected either. Hematoxylin-eosin staining and immunohistochemical staining revealed xenografts on the convex surfaces of the brains of mice that underwent CD133+ astrocytoma transplantation. Some sporadic astroglioma cells showed pseudopodium-like structures, which extended into the cerebral white matter. However, it should be emphasized that the subcortex xenograft with Notch-immunopositive expression was found in the fourth mouse received injection of CD133- astrocytoma cells. However, these findings suggest that the Notch pathway plays an important role in the formation of astrocytomas, and can be considered a novel treatment target for diffuse spinal cord astrocytoma.     

The pathological characteristics of glioma stem cell niches

Brain tumor stem cells (BTSC) are predicted to be critical drivers of tumor progression due to their self-renewal capacity and limitless proliferative potential. Recent studies suggest that stem cells are controlled by a particular microenvironment known as a “niche”. We therefore analysed human glioma tissues and found that the CD133⁺ and nestin⁺ niches are perivascularly localized in all glioma tissues. Furthermore, there is a positive correlation between the CD133⁺ niches and CD133⁺ blood vessels, which is similar to the correlation between the nestin⁺ niches and nestin⁺ blood vessels. We demonstrate that both CD133⁺ blood vessels and nestin⁺ blood vessels have an important role in maintaining the structure of the glioma stem cell niche. Moreover, the abundance of CD133⁺ niches and nestin⁺ niches increases significantly as tumor grade increases. These findings provide a new insight into the biology of BTSC and open a new perspective for targeted therapy against the brain tumors.     

Mobilization of CD133+CD34− cells in healthy individuals following whole‐body acupuncture for spinal cord injuries

Acupuncture can alleviate symptoms of spinal cord injuries (SCI). The underlying mechanism, however, is unknown. We hypothesized that stem cells could be mobilized by acupuncture. Therefore, we enrolled 14 healthy study participants using acupuncture points for the treatment of SCI. The frequency of CD133 and CD34 cells in peripheral blood and the serum concentrations of matrix metalloproteinase (MMP)‐9, brain‐derived neurotrophic factor (BDNF), nerve growth factor (NGF), and interleukin‐6 were determined before and after acupuncture (<1 hr, 24 hr, and 48 hr). CD133⁺34^? cells were doubled 48 hr after acupuncture, with concomitant decreases in BDNF and MMP‐9 levels. Interleukin‐6 remained below detectable levels, eliminating a stress‐induced cell release. Individuals acupunctured on control counterpoints showed no changes in CD133⁺ cells. Our results indicate that acupuncture for SCI can mobilize human CD133⁺34^? cells. © 2009 Wiley‐Liss, Inc.     

Neuroimmune processes associated with Wallerian degeneration support neurotrophin‐3‐induced axonal sprouting in the injured spinal cord

Lesions of the spinal cord cause two distinctive types of neuroimmune responses, a response at the lesion site that leads to additional tissue destruction and a more subtle response, termed Wallerian degeneration (WD), that occurs distal to the lesion site. We have evidence that the neuroimmune response associated with WD may support tissue repair. Previously, we found that overexpression of neurotrophin‐3 (NT‐3) induced axonal growth in the spinal cord after a unilateral corticospinal tract (CST) lesion, but only if the immune system was intact and activated. We reasoned that a neuroimmune response associated with WD was involved in this neuroplasticity. To test this, we compared NT‐3‐induced axonal sprouting in athymic nude rats that lack functional T cells with rats with functional T cells and in nude rats grafted with CD4⁺ T cells or CD8⁺ T cells. There was no sprouting in nude rats and in nude rats grafted with CD8⁺ T cells. However, nude rats grafted with CD4⁺ T cells mounted a sprouting response. To determine which CD4⁺ subtype, type 1 T helper (Th1) or type 2 T helper (Th2) cells, was responsible, we grafted Th1 and Th2 cells into nude rats and tested whether they would support sprouting. Axonal sprouting was greater in rats grafted with Th2 cells, demonstrating that the Th2 subtype was responsible for supporting axonal sprouting. These data suggest that WD activates Th2 cells that, along with the direct effects of NT‐3 on CST axons, act to support axonal sprouting in the lesioned spinal cord. © 2013 Wiley Periodicals, Inc.     

Effects of human umbilical cord blood CD34+ cell transplantation in neonatal hypoxic-ischemia rat model

Perinatal brain injury can cause death in the neonatal period and lifelong neurodevelopmental deficits. Stem cell transplantation had been proved to be effective approach to ameliorate neurological deficits after brain damage. In this study we examine the effect of human umbilical cord blood CD34⁺ cells on model of neonatal rat hypoxic–ischemic brain damage and compared the neuroprotection of transplantation of CD34⁺ cells to mononuclear cells from which CD34⁺ cells isolated on neonatal hypoxic-ischemia rat model. Seven-day-old Sprague-Dawley rats were subjected to hypoxic-ischemic (HI) injury, CD34⁺ cells (1.5?×?10⁴?cells) or mononuclear cells (1.0?×?10⁶?cells) were transplanted into mice by tail vein on the 7?day after HI. The transplantation of CD34⁺ cells significantly improved motor function of rat, and reduced cerebral atrophy, inhibited the expression of glial fibrillary acidic protein (GFAP) and apoptosis-related genes: TNF-α, TNFR1, TNFR2, CD40, Fas, and decreased the activation of Nuclear factor kappa B (NF-κB) in damaged brain. CD34⁺ cells treatment increased the expression of DCX and lectin in ipsilateral brain. Moreover, the transplantation of CD34⁺ cells and MNCs which were obtained from the same amount of human umbilical cord blood had similar effects on HI. Our data demonstrated that transplantation of human umbilical cord blood CD34+ cells can ameliorate the neural functional defect and reduce apoptosis and promote nerve and vascular regeneration in rat brain after HI injury and the effects of transplantation of CD34+ cells were comparable to that of MNCs in neonatal hypoxic-ischemia rat model.     

MiR-29a-Mediated CD133 Expression Contributes to Cisplatin Resistance in CD133<Superscript>+</Superscript> Glioblastoma Stem Cells

CD133 positive (CD133⁺) cells are cancer stem cells in glioblastoma that are associated with poor prognosis and resistance to radiotherapy. However, the role of CD133 in chemoresistance is inconclusive, although recent studies suggest that increased CD133 expression may lead to increased cisplatin resistance under certain circumstances. In this study, we further explored the mechanism underlying CD133-mediated cisplatin resistance in glioblastoma stem cells. We sorted human glioblastoma T98G and U87MG cells into CD133⁺ and CD133^? pools and measured apoptosis and CD133 expression levels in response to cisplatin treatment. We predicted candidate microRNAs that might target CD133 and assessed their levels in cisplatin-treated CD133⁺ cells. Finally, we overexpressed miR-29a in CD133⁺ cells and tested its effects in cisplatin-mediated apoptosis and survival of CD133⁺ tumor bearing mice receiving cisplatin treatment. We found that CD133⁺ glioblastoma stem cells showed more resistance to cisplatin treatment. Cisplatin increased CD133 expression by suppressing miR-29a levels. MiR-29a overexpression improved sensitivity of cisplatin in CD133⁺ cells and significantly suppressed tumor growth in CD133⁺ tumor bearing mice in response to cisplatin treatment. Our data show that miR-29a ameliorates CD133-mediated chemoresistance in glioblastoma stem cells, suggesting it as a potential therapeutic target for treating glioblastoma.     

Neurochemical evidence that 3-methylglutaric acid inhibits synaptic Na,K-ATPase activity probably through oxidative damage in brain cortex of young rats

3-Methylglutaconic aciduria (MGTA) comprehends a group of disorders biochemically characterized by accumulation of 3-methylglutaric acid (MGA), 3-methylglutaconic acid (MGT) and occasionally 3-hydroxyisovaleric acid (OHIVA). Although neurological symptoms are common in the affected individuals, the mechanisms of brain damage are poorly known. In the present study we investigated the in vitro effect MGA, MGT and OHIVA, at concentrations ranging from 0.1 to 5.0 mM, on bioenergetics and oxidative stress in synaptosomal preparations isolated from cerebral cortex of young rats. MGA significantly reduced mitochondrial redox potential (25%), as determined by resazurin reduction, and inhibited the activity of Na⁺,K⁺-ATPase (30%), whereas MGT and OHIVA did not modify these parameters. Moreover, the inhibitory effect elicited by MGA on Na⁺,K⁺-ATPase activity was totally prevented by co-incubation with the scavenging antioxidants creatine and melatonin, implying a role for reactive species in this effect. MGA also increased 2′,7′-dichlorofluorescein (DCFH) oxidation (30%), reinforcing that this organic acid induces reactive species production. The present data indicate that MGA compromises mitochondrial function, elicits reactive species production and inhibits the activity of a crucial enzyme implicated in neurotransmission. It is therefore presumed that these deleterious effects may play a role in the pathophysiology of the brain damage observed in patients affected by disorders in which MGA accumulates.     

Leukocyte infiltration into spinal cord of EAE mice is attenuated by removal of endothelial leptin signaling

Leptin, a pleiotropic adipokine, crosses the blood-brain barrier (BBB) and blood–spinal cord barrier (BSCB) from the periphery and facilitates experimental autoimmune encephalomyelitis (EAE). EAE induces dynamic changes of leptin receptors in enriched brain and spinal cord microvessels, leading to further questions about the potential roles of endothelial leptin signaling in EAE progression. In endothelial leptin receptor specific knockout (ELKO) mice, there were lower EAE behavioral scores in the early phase of the disorder, better preserved BSCB function shown by reduced uptake of sodium fluorescein and leukocyte infiltration into the spinal cord. Flow cytometry showed that the ELKO mutation decreased the number of CD3 and CD45 cells in the spinal cord, although immune cell profiles in peripheral organs were unchanged. Not only were CD4⁺ and CD8⁺ T lymphocytes reduced, there were also lower numbers of CD11b⁺Gr1⁺ granulocytes in the spinal cord of ELKO mice. In enriched microvessels from the spinal cord of the ELKO mice, the decreased expression of mRNAs for a few tight junction proteins was less pronounced in ELKO than WT mice, as was the elevation of mRNA for CCL5, CXCL9, IFN-γ, and TNF-α. Altogether, ELKO mice show reduced inflammation at the level of the BSCB, less leukocyte infiltration, and better preserved tight junction protein expression and BBB function than WT mice after EAE. Although leptin concentrations were high in ELKO mice and microvascular leptin receptors show an initial elevation before inhibition during the course of EAE, removal of leptin signaling helped to reduce disease burden. We conclude that endothelial leptin signaling exacerbates BBB dysfunction to worsen EAE.     

Neural progenitor cells promote corticospinal axon growth in organotypic co-cultures

The CNS is limited in regeneration following injury in adult mammals. Recent studies show that the transplantation of the neuronal progenitor cells is useful in promoting regeneration. However, the mechanisms of action of the transplanted neural progenitor cells have not been clarified. In this study, we used organotypic co-cultures with neonatal brain cortex and spinal cord as an in vitro assay system for assessing the factors that regulate corticospinal axonal growth. Our results show that the transplantation of neural progenitor cells enhanced corticospinal axon growth in these co-cultures. In addition, neural progenitor cell conditioned medium also significantly promoted axonal growth. These findings strongly suggest that factors derived from neural progenitor cells participate in the effect on axonal growth.     

Distribution of glycinergic neurons in the brain of glycine transporter‐2 transgenic Tg(glyt2:Gfp) adult zebrafish: Relationship to brain–spinal descending systems

We used a Tg(glyt2:gfp) transgenic zebrafish expressing the green fluorescent protein (GFP) under control of the glycine transporter 2 (GLYT2) regulatory sequences to study for the first time the glycinergic neurons in the brain of an adult teleost. We also performed in situ hybridization using a GLYT2 probe and glycine immunohistochemistry. This study was combined with biocytin tract tracing from the spinal cord to reveal descending glycinergic pathways. A few groups of GFP⁺/GLYT2^? cells were observed in the midbrain and forebrain, including numerous pinealocytes. Conversely, a small nucleus of the midbrain tegmentum was GLYT2⁺ but GFP^?. Most of the GFP⁺ and GLYT2⁺ neurons were observed in the rhombencephalon and spinal cord, and a portion of these cells showed double GLYT2/GFP labeling. In the hindbrain, GFP/GLYT2⁺ populations were observed in the medial octavolateral nucleus; the secondary, magnocellular, and descending octaval nuclei; the viscerosensory lobes; and reticular populations distributed from trigeminal to vagal levels. No glycinergic cells were observed in the cerebellum. Tract tracing revealed three conspicuous pairs of GFP/GLYT2⁺ reticular neurons projecting to the spinal cord. In the spinal cord, GFP/GLYT2⁺ cells were observed in the dorsal and ventral horns. GFP⁺ fibers were observed from the olfactory bulbs to the spinal cord, although their density varied among regions. The Mauthner neurons received very rich GFP⁺ innervation, mainly around the axon cap. Comparison of the zebrafish glycinergic system with the glycinergic systems of other adult vertebrates reveals shared patterns but also divergent traits in the evolution of this system. J. Comp. Neurol. 521:389–425, 2013. © 2012 Wiley Periodicals, Inc.     

Presence of pluripotent CD133+ cells correlates with malignancy of gliomas

BackgroundPresence of CD133⁺ cancer stem cells has been demonstrated within glioblastoma multiforme (GBM), the most malignant phenotype of gliomas (WHO grade IV). Since GBM frequently develops from low grade gliomas (WHO grade II) we assessed a possible qualitative or quantitative correlation of CD133⁺ cells and glioma grade to get new insights in gliomagenesis.ResultsThe amount of CD133⁺ cells within the bulk tumor mass, analyzed by immunostaining and Western blotting, showed a clear quantitative correlation with glioma grade (WHO° II, III and IV). Most of CD133⁺ cells were arranged in clusters frequently associated to tumor vessels. Protein analysis revealed high cellular coexpression of CD133 with Musashi-I but not CD34 indicating a neural, i.e. local origin of these cells. In vitro, no differences in stem cell properties concerning self-renewal and multi-lineage differentiation have been found for CD133⁺ cells isolated from gliomas of different grades.ConclusionsThese findings indicate a solely quantitative correlation of glioma grade with the presence of neural CD133⁺ cells within tumors supporting the concept of a CD133⁺ stem cell dependent gliomagenesis.     

Corticosteroid treatment of experimental autoimmune encephalomyelitis in the Lewis rat results in loss of Vβ8.2 and myelin basic protein-reactive cells from the spinal cord, with increased total T-cell apoptosis but reduced apoptosis of Vβ8.2 cells

We have studied the effects of corticosteroid treatment on the numbers of lymphocytes obtained from the spinal cords of Lewis rats with acute experimental autoimmune encephalomyelitis (EAE) induced by inoculation with myelin basic protein (MBP) and adjuvants. Flow cytometric studies showed that treatment with dexamethasone (4 mg/kg) 8–12 h prior to study on day 14 after inoculation resulted in a reduction in the numbers of CD5⁺, TCRαβ⁺ and Vβ8.2⁺ cells in the spinal cord. Limiting dilution analysis indicated that dexamethasone treatment 12 h prior to study on day 12 after inoculation reduced the frequencies of MBP-reactive and interleukin-2-responsive lymphocytes in the spinal cord to low levels, but reduced the frequency of concanavalin-A-responsive lymphocytes to a lesser extent. Using propidium iodide staining of nuclear chromatin we also studied lymphocyte apoptosis. Greater numbers of apoptotic cells were found in the cells extracted from the spinal cords of rats, examined on day 14, that had been treated 1–12 h previously with dexamethasone, than in saline-treated controls. This increased level of apoptosis was observed in the CD5⁺ and TCRαβ⁺ cell populations. At 1–4 h after dexamethasone treatment there was a reduction in the selective apoptosis of Vβ8.2⁺ cells that normally occurs during spontaneous recovery from EAE. Therefore apoptosis of Vβ38.2⁺ cells cannot explain the reduction in the numbers of Vβ8.2⁺ cells and MBP-reactive cells in the CNS after dexamethasone treatment. By 8–12 h after dexamethasone treatment the selectivity of the apoptotic process was restored. These studies suggest that a reduction in the number of T-lymphocytes in the central nervous system contributes to the beneficial effects of corticosteroids in EAE.     

Blood vessels expressing CD90 in human and rat brain tumors

Blood vessels in brain tumors, particularly glioblastomas, have been shown to express CD90. CD90⁺ cells in and around blood vessels in cancers including brain tumors have been identified as endothelial cells, cancer stem cells, fibroblasts or pericytes. In this study, we aimed to determine the nature or type(s) of cells that express CD90 in human brain tumors as well as an experimental rat glioma model by double immunofluorescence staining. The majority of CD90⁺ cells in human glioblastoma tissue expressed CD31, CD34 and von Willebrand factor, suggesting that they were endothelial cells. Vasculatures in a metastatic brain tumor and meningioma also expressed CD90. CD90⁺ cells often formed glomeruloid structures, typical of angiogenesis in malignant tumors, not only in glioblastoma but also in metastatic tumors. Some cells in the middle and outer layers of the vasculatures expressed CD90. Similar results were obtained in the rat glioma model. There were cells expressing both α‐smooth muscle actin and CD90 in the middle layer of blood vessels, indicating that smooth muscle cells and/or pericytes may express CD90. CD90⁺ vasculatures were surrounded by tumor‐associated macrophages (TAMs). Thus, in addition to endothelial cells, some other types of cells, such as smooth muscle cells, pericytes and fibroblasts constituting the vasculature walls in brain tumors expressed CD90. Because CD90 has been shown to interact with integrins expressed by circulating monocytes, CD90 might be involved in angiogenesis through recruitment and functional regulation of TAMs in tumors. CD90⁺ vasculatures may also interact with tumor cells through interactions with integrins. Because CD90 was not expressed by vasculatures in normal brain tissue, it might be a possible therapeutic target to suppress angiogenesis and tumor growth.     

Inhibition of phosphorylated STAT3 by cucurbitacin I enhances chemoradiosensitivity in medulloblastoma-derived cancer stem cells

Introduction

CD133 (PROM1) is a potential marker for cancer stem cells (CSCs), including those found in brain tumors. Recently, medulloblastoma (MB)-derived CD133-positive cells were found to have CSC-like properties and were proposed to be important contributors to tumorigenicity, cancer progression, and chemoradioresistance. However, the biomolecular pathways and therapeutic targets specific to MB-derived CSCs remain unresolved.

Materials and methods

In the present study, we isolated CD133⁺ cells from MB cell lines and determined that they showed increased tumorigenicity, radioresistance, and higher expression of both embryonic stem cell-related and drug resistance-related genes compared to CD133^? cells. Bioinformatics analysis suggested that the STAT3 pathway might be important in MB and CD133⁺ cells. To evaluate the effects of inhibiting the STAT3 pathway, MB-derived CD133^+/? cells were treated with the potent STAT3 inhibitor, cucurbitacin I. Treatment with cucurbitacin I significantly suppressed the CSC-like properties and stemness gene signature of MB-derived CD133⁺ cells. Furthermore, cucurbitacin I treatment increased the apoptotic sensitivity of MB-derived CD133⁺ cells to radiation and chemotherapeutic drugs. Notably, cucurbitacin I demonstrated synergistic effects with ionizing radiation to inhibit tumorigenicity in MB-CD133⁺-inoculated mice.

Results

These results indicate that the STAT3 pathway plays a key role in mediating CSC properties in MB-derived CD133⁺ cells. Targeting STAT3 with cucurbitacin I may therefore represent a novel therapeutic approach for treating malignant brain tumors.     

Lymphokine mRNA expression in the spinal cords of Lewis rats with experimental autoimmune encephalomyelitis is associated with a host recruited CD45R hi/CD4 population during recovery

To evaluate CD4⁺ T cell subpopulations involved in the induction and recovery from experimental autoimmune encephalomyelitis (EAE), the CD45R phenotype and lymphokine mRNA profile was evaluated for encephalitogenic CD4⁺ T cell lines in vitro and compared to CD4^* T cells islated from the spinal cord of Lewis rats with EAE were > 90% of the myelin basic protein (MBP)-specific T cell lines and clones that adoptively transferred EAE were > 90% CD4⁺ and > 90% CD45R lo. A time course of EAE disease progression was monitored as a function of the percentage of CD45R hi/CD4⁺ T cells isolated from the spinal cords of diseased animals. The majority of CD4⁺ T cells found in the central nervous system during the early phase of passive EAE were CD45R lo (the same as the encephalitogenic lines/clones). A large increase of the CD45R hi/CD4⁺ T cells (up to 45%) was observed during the peak and recovery phases of EAE. Lymphokine mRNA production was analyzed from antigen-stimulated MBP-specific lines, and from spinal cord lymphocytes isolated from rats with EAE. The BP-specific lines produced Th1 lymphokines (IL-2, IFN-γ, and TNF-α), while the spinal cord lymphocytes produced the same Th1 lymphokines as well as IL-4 and IL-10. The CD45R hi/CD4⁺ T cells isolated from the spinal cords were larger and expressed more lymphokine RNA per cell than the CD45R lo/CD4⁺ T cells. The encephalitogenic cells (CD45R hi/CD4⁺ T detected in the spinal cords of rats with a fluorescent dye and by allelic transfers and all of the CD45R hi/CD4⁺ lymphocytes found in the spinal cells were found to be host recruited. Thus it appears that the CD45R hi/CD4⁺ lymphocytes found in the spinal cord represent a host-recruited, activated cellular infiltrate that increased in number in the recovery phase of EAE and synthesized both Th1 and Th2 lymphokines.     

Gintonin mitigates experimental autoimmune encephalomyelitis by stabilization of Nrf2 signaling via stimulation of lysophosphatidic acid receptors

Gintonin (GT), a glycolipoprotein fraction isolated from ginseng, exerts neuroprotective effects in models of neurodegenerative diseases such as Alzheimer’s disease. However, the in vivo role of GT in multiple sclerosis (MS) has not been clearly resolved. We investigated the effect of GT in myelin oligodendrocyte glycoprotein (MOG_35-55)-induced experimental autoimmune encephalomyelitis (EAE), an animal model of MS. GT alleviated behavioral symptoms of EAE associated with reduced demyelination, diminished infiltration and activation of immune cells (microglia and macrophage), and decreased expression of inflammatory mediators in the spinal cord of the EAE group compared to that of the sham group. GT reduced the percentages of CD4⁺/IFN-γ⁺ (Th1) and CD4⁺/IL-17⁺ (Th17) cells but increased the population of CD4⁺/CD25⁺/Foxp3⁺ (Treg) cells in the spinal cord, in agreement with altered mRNA expression of IFN-γ, IL-17, and TGF-ß in the spinal cord in concordance with mitigated blood–brain barrier disruption. The underlying mechanism is related to inhibition of the ERK and p38 mitogen-activated protein kinases (MAPKs) and nuclear factor-kappa B (NF-κB) pathways and the stabilization of nuclear factor erythroid 2-related factor 2 (Nrf2) via increased expression of lysophosphatidic acid receptor (LPAR) 1–3. Impressively, these beneficial effects of GT were completely neutralized by inhibiting LPARs with Ki16425, a LPAR1/3 antagonist. Our results strongly suggest that GT may be able to alleviate EAE due to its anti-inflammatory and antioxidant activities through LPARs. Therefore, GT is a potential therapeutic option for treating autoimmune disorders including MS.     

Autoradiographic demonstration of the central origin of regenerating fibers into iris tissue implants in the rat mesencephalon

One model useful for demonstrating axonal regeneration in the central nervous system is that in which peripheral tissue, particularly the iris, is implanted into the brain or spinal cord. The aim of the present study was to establish the origin of some of the regenerating fibers by the use of an autoradiographic tracing technique. Briefly, iris implants were inserted in the caudal mesencephalon in the region of the dorsal tegmental bundle in unilaterally sympathectomized rats. Approximately two weeks later, subjects were injected with [³H]leucine in the region of the ipsilateral locus coeruleus and sacrificed two days later. Numerous labeled fibers were observed within the implants and traversing between brain and implant. Fibers formed loose plexuses, coursed along smooth muscle and grew toward blood vessels within the implants. These results may be added to fluorescent histochemical, biochemical and ultrastructural evidence supporting the regenerative capacity of central noradrenergic fibers.     

MHC class II antigen expression and T-cell infiltration in the demyelinating CNS and PNS of the twitcher mouse

The expression of the major histocompatibility complex class II antigens (Ia) was investigated in the central and peripheral nervous systems of the twitcher mouse, an authentic murine model of globoid cell leukodystrophy (Krabbe disease) in humans. In this mutant, demyelination is noted as early as postnatal day 10 in the peripheral nerve and day 20 in the spinal cord. Emergence of Ia antigen expressing cells (Ia⁺ cells) was largely coincident with the initiation of demyelination, suggesting the importance of local factors for the induction of Ia antigens. Ia⁺ cells gradually increased in number with the progression of demyelination, but reached a plateau between postnatal days 30 and 40, then rapidly decreased despite continuous demyelination in both central and peripheral nervous systems. The only exception was in the spinal cord where Ia⁺ cells were numerous even at postnatal day 50. The cells expressing L3T4 antigen (L3T4⁺ cells), which is a mouse CD4 antigen, and the interleukin-2 receptor antigen expressing cells (IL-2R⁺ cells), also appeared in the regions where Ia⁺ cells were observed. L3T4⁺ cells were still abundant after P45, when Ia⁺ and IL-2R⁺ cells decreased. Combined autoradiographic and immunocytochemical studies demonstrated mitotic activity in a few Ia⁺ cells. However, low labeling indices of Ia⁺ cells in both cerebrum and sciatic nerve suggest that the increase of Ia⁺ cells was largely due to either induction of Ia antigens on local cells or new recruitment of Ia⁺ cells from the circulation. Expression of Ia antigens associated with an emergence of L3T4⁺ or IL-2R⁺ cells in the demyelinating lesions may indicate involvement of immunological responses in the pathogenesis of this metabolic demyelinating disorder. Alternatively, these apparent immunological phenomena may be tentative and non-specific local reactions to degenerating tissue components such as myelin. The mechanism(s) regulating the expression of Ia antigens in this genetic metabolic demyelinating disease is yet to be determined.     

Hydrogels Containing Peptide or Aminosugar Sequences Implanted into the Rat Brain: Influence on Cellular Migration and Axonal Growth

Biocompatible polymer matrices for implantation into lesion sites in the brain were synthesized by incorporating peptide or aminosugar sequences intoN-(2-hydroxypropyl)methacrylamide (HPMA) hydrogels. RGD peptide sequences were chemically linked to the hydrogel backbone via a glycylglycine spacer; aminosugars were glucosamine (NHGlc) orN-acetylglucosamine residues. Unmodified or sequence containing HPMA hydrogels were implanted into the lesioned optic tract or cerebral cortex of juvenile (17- to 19-day-old) or adult rat brains, respectively. After 10–12 months host animals were perfused and the brains were processed for immunohistochemistry using antibodies to neurofilaments (RT97), laminin, glial fibrillary acidic protein (GFAP), carbonic anhydrase II (CAII), S100 protein, macrophages (ED1), and myelin basic protein (MBP). Unmodified (control) HPMA hydrogels contained no cellular infiltration or axonal growth. Peptide (RGD)- and aminosugar-modified hydrogels showed increased adhesion properties with host neural tissue, were vascularized, and were infiltrated by host nonneuronal cells. Astrocytes (GFAP⁺) and macrophages (ED1⁺) were the major cell types seen within modified HPMA hydrogels, the largest numbers being found in RGD-containing polymers. CAII⁺oligodendroglia were not seen within any of the hydrogel matrices. RT97⁺/MBP⁻axons grew into both the RGD and NHGlc hydrogel matrices for small distances. The number of axons was greatest in hydrogels implanted into cerebral cortex but in both cortex and optic tract implants the highest density of axons was seen in polymers containing RGD. The findings of this study are discussed in the context of CNS tissue replacement and the construction of bioactive scaffolds to promote regenerative axonal growth across areas of injury in the brain and spinal cord.