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Neuronal regulation of diverse physiological functions requires complex molecular interactions in innervated tissues to maintain proper organ function. Here we show that loss of the neuronal cell surface adhesion/recognition molecule Contactin-1 (Cntn1) directly impairs intestinal function causing wasting that subsequently results in global immune defects. Loss of Cntn1 results in hematologic alterations and changes in blood metabolites associated with malnourishment. We found thymus and spleen of Cntn1-deficient animals atrophied with severe reductions in lymphocyte populations. Elevated thymic Gilz expression indicated ongoing glucocorticoid signaling in Cntn1-deficient animals, consistent with the malnourishment phenotype. Intestinal Contactin-1 was localized to neurons in the villi and the submucosal/myenteric plexus that innervates smooth muscle. Loss of Cntn1 was associated with reduced intestinal Bdnf and Adrb2, indicating reduced neuromuscular crosstalk. Additionally, loss of Cntn1 resulted in reduced recruitment of CD3+ T cells to villi within the small intestine. Together, these data illustrate the critical role of Contactin-1 function within the gut, and how this is required for normal systemic immune functions.

B cell development is a highly regulated process involving multiple differentiation steps, yet many details regarding this pathway remain unknown. Sequencing of patients with B cell-restricted immunodeficiency reveals autosomal dominant mutations in TOP2B. TOP2B encodes a type II topoisomerase, an essential gene required to alleviate topological stress during DNA replication and gene transcription, with no previously known role in B cell development. We use Saccharomyces cerevisiae, and knockin and knockout murine models, to demonstrate that patient mutations in TOP2B have a dominant negative effect on enzyme function, resulting in defective proliferation, survival of B-2 cells, causing a block in B cell development, and impair humoral function in response to immunization., Topoisomerases are required to release topological stress on DNA during replication and transcription. Here, Broderick et al. report genetic variants in TOP2B that cause a syndromic B cell immunodeficiency associated with reduced TOP2B function, defects in B cell development and B cell activation.

The NG2 proteoglycan promotes tumor growth as a component of both tumor and stromal cells. Using intracranial, NG2-negative B16F10 melanomas, we have investigated the importance of pericyte and macrophage NG2 in brain tumor progression. Reduced melanoma growth in myeloid-specific NG2 null (Mac-NG2ko) and pericyte-specific NG2 null (PC-NG2ko) mice demonstrates the importance of NG2 in both stromal compartments. In each genotype, loss of pericyte-endothelial cell interaction diminishes formation of endothelial junctions and assembly of the basal lamina. Tumor vessels in Mac-NG2ko mice have smaller diameters, reduced patency, and increased leakiness compared to PC-NG2ko mice, thus decreasing tumor blood supply and increasing hypoxia. While reduced pericyte interaction with endothelial cells in PC-NG2ko mice results from loss of pericyte activation of β1 integrin signaling in endothelial cells, reduced pericyte-endothelial cell interaction in Mac-NG2ko mice results from 90% reduced macrophage recruitment. The absence of macrophage-derived signals in Mac-NG2ko mice causes loss of pericyte association with endothelial cells. Reduced macrophage recruitment may be due to diminished activation of integrins in the absence of NG2, causing decreased macrophage interaction with endothelial adhesion molecules that are needed for extravasation. These results reflect the complex interplay that occurs between macrophages, pericytes, and endothelial cells during tumor vascularization.

Macrophage infiltration is a factor in most if not all inflammatory pathologies. Understanding molecular interactions that underlie this process is therefore important for our ability to modulate macrophage behavior for therapeutic purposes. Our studies show that cell surface expression of the nerveglial antigen 2 (NG2) proteoglycan is important for the ability of macrophages to colonize both brain tumors and sites of central nervous system (CNS) demyelination. Myeloid-specific ablation of NG2 using LysM-Cre deleter mice results in large decreases in macrophage abundance in both an intracranial melanoma model and a lysolecithin model of spinal cord demyelination. In the melanoma model, decreased macrophage recruitment in the NG2 null mice leads to diminished tumor growth. In line with observations in the literature, this phenomenon is based in part on deficits in tumor vascularization that result from loss of pericyte interaction with endothelial cells in the absence of a macrophage-derived factor(s). In the demyelination model, decreased macrophage infiltration in the NG2 null mice is associated with an initial reduction in lesion size, but nevertheless also with deficits in repair of the lesion. Diminished myelin repair is due not only to reduced clearance of myelin debris, but also to decreased proliferation/recruitment of oligodendrocyte progenitor cells in the absence of a macrophage-derived factor(s). Thus, in both models macrophages have secondary effects on other cell types that are important for progression of the specific pathology. Efforts are underway to identify mechanisms by which NG2 influences macrophage recruitment and by which macrophages signal to other cell types involved in the pathologies.

Guanylyl cyclase (GC) as the effector molecule for nitric oxide (NO) plays a key role in the NO/cGMP signalling cascade. Based on these observations, our study focused on NO/sGC signalization in the bulbospinal respiratory pathway. The distribution of neuronal nitric oxide synthase (nNOS), β1 subunit of soluble guanylyl cyclase (β1sGC) and synaptophysin (SYN) was explored in the upper part of the respiratory pathway after C2-C3 hemisection of the spinal cord in male Wistar rats. Unilateral injection of Fluorogold into the phrenic nucleus (PN) at C4 level and survival of animals for 2 days revealed many Fluorogold fluorescent neurons in the ventral respiratory group (VRG) of the medulla, mostly on the contralateral side. Under physiological conditions we noted nNOS-fluorescent terminals of VRG neurons around β1sGC fluorescent motoneurons in the PN. A strong depletion of nNOS/SYN fluorescent terminals was noted 8 days after hemisection around alpha motoneurons in the PN on the contralateral side. On the side of injury, nNOS/SYN fluorescent puncta were detected around phrenic motoneurons only sporadically. Phrenic alpha motoneurons responded to C2-C3 hemisection by a loss of β1sGC positivity. The results confirm, that β1sGC immunoreactive phrenic motoneurons are innervated by nNOS positive terminals coming from the VRG.

The NG2 proteoglycan has been shown to promote proliferation and motility in a variety of cell types. The presence of NG2 on oligodendrocyte progenitor cells (OPCs) suggests that the proteoglycan may be a factor in expansion of the OPC pool to fill the entire central nervous system prior to OPC differentiation to form myelinating oligodendrocytes. Comparisons of postnatal cerebellar myelination in wild type and NG2 null mice reveal reduced numbers of OPCs in developing white matter of the NG2 null mouse. Quantification of BrdU incorporation shows that reduced proliferation is a key reason for this OPC shortage, with the peak of OPC proliferation delayed by 4-5 days in the absence of NG2. As a result of the subnormal pool of OPCs, there is also a delay in production of mature oligodendrocytes and myelinating processes in the NG2 null cerebellum. NG2 may promote OPC proliferation via enhancement of growth factor signaling or mediation of OPC interaction with unmyelinated axons.

Using a rat model of ischemic paraplegia, we examined the expression of spinal AMPA receptors and their role in mediating spasticity and rigidity. Spinal ischemia was induced by transient occlusion of the descending aorta combined with systemic hypotension. Spasticity/rigidity were identified by simultaneous measurements of peripheral muscle resistance (PMR) and electromyography (EMG) before and during ankle flexion. In addition, Hoffman reflex (H-reflex) and motor evoked potentials (MEPs) were recorded from the gastrocnemius muscle. Animals were implanted with intrathecal catheters for drug delivery and injected with the AMPA receptor antagonist NGX424 (tezampanel), glutamate receptor 1 (GluR1) antisense, or vehicle. Where intrathecal vehicle had no effect, intrathecal NGX424 produced a dose-dependent suppression of PMR [ED50of 0.44 μg (0.33–0.58)], as well as tonic and ankle flexion-evoked EMG activity. Similar suppression of MEP and H-reflex were also seen. Western blot analyses of lumbar spinal cord tissue from spastic animals showed a significant increase in GluR1 but decreased GluR2 and GluR4 proteins. Confocal and electron microscopic analyses of spinal cord sections from spastic animals revealed increased GluR1 immunoreactivity in reactive astrocytes. Selective GluR1 knockdown by intrathecal antisense treatment resulted in a potent reduction of spasticiy and rigidity and concurrent downregulation of neuronal/astrocytic GluR1 in the lumbar spinal cord. Treatment of rat astrocyte cultures with AMPA led to dose-dependent glutamate release, an effect blocked by NGX424. These data suggest that an AMPA/kainate receptor antagonist can represent a novel therapy in modulating spasticity/rigidity of spinal origin and that astrocytes may be a potential target for such treatment.

Early stage growth of intracranial B16F10 tumors is reduced by 87% in myeloid-specific NG2 null (Mac-NG2ko) mice and by 77% in pericyte-specific NG2 null (PC-NG2ko) mice, demonstrating the importance of the NG2 proteoglycan in each of these stromal compartments. In both genotypes, loss of pericyte-endothelial cell interaction results in numerous structural defects in tumor blood vessels, including decreased formation of endothelial cell junctions and decreased assembly of the vascular basal lamina. All vascular deficits are larger in Mac-NG2ko mice than in PC-NG2ko mice, correlating with the greater decrease in pericyte-endothelial cell interaction in Mac-NG2ko animals. Accordingly, tumor vessels in Mac-NG2ko mice have a smaller diameter, lower degree of patency, and higher degree of leakiness than tumor vessels in PC-NG2ko mice, leading to less efficient tumor blood flow and to increased intratumoral hypoxia. While reduced pericyte interaction with endothelial cells in PC-NG2ko mice is caused by loss of NG2-dependent pericyte activation of β1 integrin signaling in endothelial cells, reduced pericyte-endothelial cell interaction in Mac-NG2ko mice is due to a 90% reduction in NG2-dependent macrophage recruitment to tumors. The absence of a macrophage-derived signal(s) in Mac-NG2ko mice results in the loss of pericyte ability to associate with endothelial cells, possibly due to reduced expression of N-cadherin by both pericytes and endothelial cells.

1. The aim of the present study was to examine the occurrence of the neuronal nitric oxide synthase immunoreactivity in the stretch reflex circuit pertaining to the quadriceps femoris muscle in the dog. 2. Immunohistochemical processing for neuronal nitric oxide synthase and histochemical staining for nicotinamide adenine dinucleotide phosphate diaphorase were used to demonstrate the presence of neuronal nitric oxide synthase in the proprioceptive afferents issuing in the quadriceps femoris muscle. The retrograde tracer Fluorogold injected into the quadriceps femoris muscle was used to detect the proprioceptive afferents and their entry into the L5 and L6 dorsal root ganglia. 3. A noticeable number of medium-sized intensely nitric oxide synthase immunolabelled somata (1000-2000 microm(2) square area) was found in control animals in the dorsolateral part of L5 and L6 dorsal root ganglia along with large-caliber intraganglionic nitric oxide synthase immunolabelled fibers, presumed to be Ia axons. Before entering the dorsal funiculus the large-caliber nitric oxide synthase immunolabelled fibers of the L5 and L6 dorsal roots formed a massive medial bundle, which upon entering the dorsal root entry zone reached the dorsolateral part of the dorsal funiculus and were distributed here in a funnel-shaped fashion. The largest nitric oxide synthase immunolabelled fibers, 8.0-9.2 microm in diameter, remained close to the dorsal horn, while medium-sized fibers were seen dispersed across the medial portion of the dorsal funiculus. Single, considerably tapered nitric oxide synthase immunolabelled fibers, 2.2-4.6 microm in diameter, were seen to proceed in ventrolateral direction until they reached the mediobasal portion of the dorsal horn and the medial part of lamina VII. In lamina IX, only short fragments of nitric oxide synthase immunoreactive fibers and their terminal ramifications could be seen. Nitric oxide synthase immunolabelled terminals varying greatly in size were identified in control material at the base of the dorsal horn, in the vicinity of motoneurons ventrally and ventrolaterally in L5 and L6 segments and in Clarke's column of L3 and L4 segments. Injections of the retrograde tracer Fluorogold into the quadriceps femoris muscle and cut femoral nerve, combined with nitric oxide synthase immunohistochemistry of the L5 and L6 dorsal root ganglia, confirmed the existence of a number of medium-sized nitric oxide synthase immunoreactive and Fluorogold-fluorescent somata presumed to be proprioceptive Ia neurons (1000-2000 microm(2) square area) in the dorsolateral part of both dorsal root ganglia. L5 and L6 dorsal rhizotomy caused a marked depletion of nitric oxide synthase immunoreactivity in the medial bundle of the L5 and L6 dorsal roots and in the dorsal funiculus of L5 and L6 segments. 4. The analysis of control material and the degeneration of the large- and medium-caliber nitric oxide synthase immunoreactive Ia fibers in the dorsal funiculus of L5 and L6 segments confirmed the presence of nitric oxide synthase in the afferent limb of the monosynaptic Ia-motoneuron stretch reflex circuit related to the quadriceps femoris muscle.

1. To test our hypothesis that a transient nonlethal ischemic insult benefits the lumbosacral spinal cord ischemic injury, nestin, the marker of proliferating cells, and Fluoro-Jade B, the marker of degenerating cells, were used in rats. Morphological outcome was evaluated after 12-min ischemia versus 12-min ischemia preconditioned by 3-min ischemic period and 30-min recirculation (IPC), in each group followed by 2, 3, and 4 days of posttreatment survival. 2. Twelve-minute ischemia, inducing nestin-positivity in ependyma and reactive astrocytes at the L(1-3) spinal cord segments, shows this region as the viable region of spinal cord in all postischemic survival periods. On the other hand, abundance of Fluoro-Jade B-positive cells, distributed throughout the dorsal horn and intermediate zone of L4-S2 segments, points out the most injured spinal cord region by ischemia. 3. After the same ischemic insult in IPC rats only a few nestin-positive ependymal cell and reactive astrocytes appeared beside the nestin-positive vessels in the lower lumbar and sacral spinal cord segments of all survival periods. The appearance of nestin-positive cells in the spinal cord segments, which "should have been affected" by ischemia indicates protection of this region by the IPC treatment. 4. The number and density evaluation of Fluoro-Jade B fluorescent cells of L4-S2 segments after ischemia and IPC confirmed that degenerating cells were significantly reduced in the IPC rats in all survival periods. 5. Our results showing the immunohistochemical response of epemdyma, committed to the presence of viable tissue, indicate that the ependymal cells may contribute to the ischemic resistance in the IPC rats.

Previous investigations from our laboratory have documented that the neuropil of the phrenic nucleus contains a dense accumulation of punctate nicotinamide adenine dinucleotide phosphate diaphorase staining. In this study we investigated the occurrence and origin of punctate nitric oxide synthase immunoreactivity in the neuropil of the phrenic nucleus in C3–C5 segments, supposed to be the terminal field of the premotor bulbospinal respiratory nitric oxide synthase-immunoreactive pathway in the dog. As the first step, nitric oxide synthase immunohistochemistry was used to characterize nitric oxide synthase-immunoreactive staining of the phrenic nucleus and nitric oxide synthase-containing neurons in the dorsal and rostral ventral respiratory group and in the Botzinger complex of the medulla. Dense punctate nitric oxide synthase immunoreactivity was found on control sections in the neuropil of the phrenic nucleus. Several thin bundles of nitric oxide synthase-immunoreactive fibers were found to enter the phrenic nucleus from the lateral and ventral column. Nitric oxide synthase-containing neurons were revealed in the dorsal respiratory group of medulla corresponding to the ventrolateral nucleus of the solitary tract and in the rostral ventral respiratory group beginning approximately 1 mm caudal to the obex and reaching to 650 μm rostral to the obex. Axotomy-induced retrograde changes, consisting in a strong upregulation of nitric oxide synthase-containing neurons, were found in the dorsal and rostral ventral respiratory group contralateral to the hemisection performed at the C2–C3 level. Concurrently, a strong depletion of the punctate nitric oxide synthase immunopositivity in the neuropil of the phrenic nucleus ipsilaterally with the hemisection was detected, thus revealing that a crossed premotor bulbospinal respiratory pathway contains a fairly high number of nitric oxide synthase-immunopositive fibers terminating in the phrenic nucleus. The use of the retrograde fluorescent tracer Fluorogold injected into the phrenic nucleus and an analysis of sections cut through the dorsal and rostral ventral respiratory group and Botzinger complex of medulla and processed for nitric oxide synthase immunocytochemistry revealed that approximately 73.8% of crossed premotor bulbospinal respiratory nitric oxide synthase-immunoreactive axons originate in the rostral ventral respiratory group and 26.2% is given by nitric oxide synthase-containing neurons of the dorsal respiratory group. A few premotor nitric oxide synthase-immunoreactive axons originating from the Botzinger complex were found. In summary, the present study provides evidence for a hitherto unknown premotor bulbospinal respiratory nitric oxide synthase-immunoreactive pathway connecting the bulbar respiratory centers with the motor neurons of the phrenic nucleus in the dog.

We used a bone marrow transplantation approach to distinguish the activities of bone marrow-derived macrophages from the activities of central nervous system-resident microglia in phenomena associated with axon demyelination and remyelination. We transplanted wild type or germline NG2 null beta-actin-EGFP expressing bone marrow into irradiated wild type or NG2 null recipient mice, followed by analysis of lysolecithin-induced spinal cord demyelination and remyelination and quantification of Iba-1+/ F4/80+/ EGFP+ macrophages and Iba-1+/ F4/80+/ EGFP- microglia. One week after microinjection of 1% lysolecithin into the spinal cord, wild type recipients receiving NG2 null bone marrow exhibit greatly reduced infiltration of macrophages into lesions, compared to wild type recipients receiving wild type bone marrow. Wild type bone marrow recipients also exhibit larger numbers of demyelinated axons than NG2 null recipients, indicative of macrophage participation in the initial myelin damage. However, wild type bone marrow recipients also exhibit superior myelin repair at 6 weeks post-injury, compared to NG2 null bone marrow recipients, demonstrating the additional importance of macrophages in remyelination. Incompletely repaired lesions in NG2 null bone marrow recipients at 6 weeks post-injury retain elevated numbers of macrophages, in contrast to lower numbers of macrophages in more completely repaired lesions in wild type bone marrow recipients. This suggests that NG2 expression renders macrophages more effective in myelin repair and less likely to promote chronic inflammation. Effective macrophage involvement in myelin repair is due in part to effects on the proliferation and/or recruitment of oligodendrocyte progenitor cells. Reduced numbers of oligodendrocyte progenitors are seen in lesions in NG2 null bone marrow recipients, likely due to deficits in macrophage production of oligodendrocyte progenitor-relevant mitogens and in phagocytosis of inhibitory myelin debris. Microglia also appear to be important for clearance of myelin debris, as indicated by reduced phagocytosis in NG2 null recipients receiving wild type bone marrow.

Background Multiple sclerosis (MS) is a demyelinating disease in which blood-derived immune cells and activated microglia damage myelin in the central nervous system. While oligodendrocyte progenitor cells (OPCs) are essential for generating oligodendrocytes for myelin repair, other cell types also participate in the damage and repair processes. The NG2 proteoglycan is expressed by OPCs, pericytes, and macrophages/microglia. In this report we investigate the effects of NG2 on these cell types during spinal cord demyelination/remyelination. Methods Demyelinated lesions were created by microinjecting 1% lysolecithin into the lumbar spinal cord. Following demyelination, NG2 expression patterns in wild type mice were studied via immunostaining. Immunolabeling was also used in wild type and NG2 null mice to compare the extent of myelin damage, the kinetics of myelin repair, and the respective responses of OPCs, pericytes, and macrophages/microglia. Cell proliferation was quantified by studies of BrdU incorporation, and cytokine expression levels were evaluated using qRT-PCR. Results The initial volume of spinal cord demyelination in wild type mice is twice as large as in NG2 null mice. However, over the ensuing 5 weeks there is a 6-fold improvement in myelination in wild type mice, versus only a 2-fold improvement in NG2 null mice. NG2 ablation also results in reduced numbers of each of the three affected cell types. BrdU incorporation studies reveal that reduced cell proliferation is an important factor underlying NG2-dependent decreases in each of the three key cell populations. In addition, NG2 ablation reduces macrophage/microglial cell migration and shifts cytokine expression from a pro-inflammatory to anti-inflammatory phenotype. Conclusions Loss of NG2 expression leads to decreased proliferation of OPCs, pericytes, and macrophages/microglia, reducing the abundance of all three cell types in demyelinated spinal cord lesions. As a result of these NG2-dependent changes, the course of demyelination and remyelination in NG2 null mice differs from that seen in wild type mice, with both myelin damage and repair being reduced in the NG2 null mouse. These studies identify NG2 as an important factor in regulating myelin processing, suggesting that therapeutic targeting of the proteoglycan might offer a means of manipulating cell behavior in demyelinating diseases.

A major limitation of neural transplantation studies is assessing the degree of host–graft interaction. In the present study, rat hippocampal/cortical embryonic neurons (E18) were infected with a lentivirus encoding enhanced green fluorescent protein (GFP) under control of the neuron-specific synapsin promoter, thus permitting robust identification of labeled neurons after in vivo grafting. Two weeks after transient forebrain ischemia or sham-surgery, GFP-expressing neurons were transplanted into CA1 hippocampal regions in immunosuppressed adult Wistar rats. The survival, distribution, phenotype, and axonal projections of GFP-immunoreactive (IR) positive transplanted neurons were evaluated in the sham-operated and ischemia-damaged CA1 hippocampal regions 4, 8, and 12 weeks after transplantation. In both experimental groups, we observed that the main phenotype of host-derived afferents projecting towards grafted GFP-IR neurons as well as transplant-derived GFP-IR efferents were glutamatergic in both animal groups. GFP axonal projections were seen in the nucleus accumbens, septal nuclei, and subiculum—known target areas of CA1 pyramidal neurons. Compared to sham-operated animals, GFP-IR neurons grafted into the ischemia-damaged CA1 migrated more extensively throughout a larger volume of host tissue, particularly in the stratum radiatum. Moreover, enhanced axonal sprouting and neuronal plasticity of grafted cells were evident in the hippocampus, subiculum, septal nuclei, and nucleus accumbens of the ischemia-damaged rats. Our study suggests that the adult rat brain retains some capacity to direct newly sprouting axons of transplanted embryonic neurons to the correct targets and that this capacity is enhanced in previously ischemia-injured forebrain.

PURPOSE: To study the microscopic structure of postmortem human Meibomian glands (MGs) in the elderly. METHODS: Human MG samples from left lower eyelids were obtained at autopsy from 5 men and 4 women with a mean age of 63.1 +/- 7.67 years. The tissues were fixed and embedded in paraffin. Serial transverse sections 5 mum thick were stained with hematoxylin and eosin (HandE), van Gieson, and Masson blue stains. Computer-assisted 3-dimensional reconstructions of MGs were performed, and morphologic and volumetric data were analyzed. RESULTS: The average length of human MGs in the nasal, central, and temporal areas was 1.551 +/- 0.43, 1.654 +/- 0.47, and 1.594 +/- 0.57 mm, respectively. The average surface area of the glands in the nasal, central, and temporal areas was 0.029 +/- 0.03, 0.033 +/- 0.01, and 0.056 +/- 0.03 mm, respectively. The average volume of glands in the nasal, central, and temporal areas was 0.054 +/- 00.4, 0.056 +/- 0.03, and 0.053 +/- 0.03 mm, respectively. A circular, floral arrangement of acini, surrounding the terminal duct just deep to the skin, is probably responsible for the circular arrangement seen clinically around each healthy orifice. We confirmed that most glands are embedded within a cylindrical, connective tissue matrix. CONCLUSIONS: We report the dimensions of normal Meibomian acini in an older population. Some structural features observed may explain normal physiologic landmarks or contribute to glandular pathophysiology.

Transient spinal cord ischemia in humans can lead to the development of permanent paraplegia with prominent spasticity and rigidity. Histopathological analyses of spinal cords in animals with ischemic spastic paraplegia show a selective loss of small inhibitory interneurons in previously ischemic segments but with a continuing presence of ventral alpha-motoneurons and descending cortico-spinal and rubro-spinal projections. The aim of the present study was to examine the effect of human spinal stem cells (hSSCs) implanted spinally in rats with fully developed ischemic paraplegia on the recovery of motor function and corresponding changes in motor evoked potentials. In addition the optimal time frame for cell grafting after ischemia and the optimal dosing of grafted cells were also studied. Spinal cord ischemia was induced for 10 min using aortic occlusion and systemic hypotension. In the functional recovery study, hSSCs (10,000-30,000 cells/0.5 mul/injection) were grafted into spinal central gray matter of L2-L5 segments at 21 days after ischemia. Animals were immunosuppressed with Prograf (1 mg/kg or 3 mg/kg) for the duration of the study. After cell grafting the recovery of motor function was assessed periodically using the Basso, Beattie and Bresnahan (BBB) scoring system and correlated with the recovery of motor evoked potentials. At predetermined times after grafting (2-12 weeks), animals were perfusion-fixed and the survival, and maturation of implanted cells were analyzed using antibodies recognizing human-specific antigens: nuclear protein (hNUMA), neural cell adhesion molecule (hMOC), neuron-specific enolase (hNSE) and synapthophysin (hSYN) as well as the non-human specific antibodies TUJ1, GFAP, GABA, GAD65 and GLYT2. After cell grafting a time-dependent improvement in motor function and suppression of spasticity and rigidity was seen and this improvement correlated with the recovery of motor evoked potentials. Immunohistochemical analysis of grafted lumbar segments at 8 and 12 weeks after grafting revealed intense hNSE immunoreactivity, an extensive axo-dendritic outgrowth as well as rostrocaudal and dorsoventral migration of implanted hNUMA-positive cells. An intense hSYN immunoreactivity was identified within the grafts and in the vicinity of persisting alpha-motoneurons. On average, 64% of hSYN terminals were GAD65 immunoreactive which corresponded to GABA immunoreactivity identified in 40-45% of hNUMA-positive grafted cells. The most robust survival of grafted cells was seen when cells were grafted 21 days after ischemia. As defined by cell survival and laminar distribution, the optimal dose of injected cells was 10,000-30,000 cells per injection. These data indicate that spinal grafting of hSSCs can represent an effective therapy for patients with spinal ischemic paraplegia.

1. Brief interruption of spinal cord blood flow resulting from transient abdominal aortic occlusion may lead to degeneration of specific spinal cord neurons and to irreversible loss of neurological function. The alteration of nitric oxide/nitric oxide synthase (NO/NOS) pool occurring after ischemic insult may play a protective or destructive role in neuronal survival of affected spinal cord segments. 2. In the present study, the spatiotemporal changes of NOS following transient ischemia were evaluated by investigating neuronal NOS immunoreactivity (nNOS-IR), reduced nicotinamide adenine dinucleotide phosphate diaphorase (NADPHd) histochemistry, and calcium-dependent NOS (cNOS) conversion of [(3)H] l-arginine to [(3)H] l-citrulline. 3. The greatest levels of these enzymes and activities were detected in the dorsal horn, which appeared to be most resistant to ischemia. In that area, the first significant increase in NADPHd staining and cNOS catalytic activity was found immediately after a 15-min ischemic insult. 4. Increases in the ventral horn were observed later (i.e., after a 24-h reperfusion period). While the most intense increase in nNOS-IR was detected in surviving motoneurons of animals with a shorter ischemic insult (13 min), the greatest increase of cNOS catalytic activity and NADPHd staining of the endothelial cells was found after stronger insult (15 min). 5. Given that the highest levels of nNOS, NADPHd, and cNOS were found in the ischemia-resistant dorsal horn, and nNOS-IR in surviving motoneurons, it is possible that NO production may play a neuroprotective role in ischemic/reperfusion injury.

1. The present study was designed to examine the nitric oxide synthase activities (constitutive and inducible) in the site of injury in response to Th10-Th11 spinal cord hemisection and, to determine whether unilateral disconnection of the spinal cord influences the NOS pools on the contra- and ipsilateral sides in segments located far away from the epicentre of injury. 2. A radioassay detection was used to determine Ca(2+)-dependent and inducible nitric oxide synthase activities. Somal, axonal and neuropil neuronal nitric oxide synthase was assessed by immunocytochemical study. A quantitative assessment of neuronal nitric oxide synthase immunoreactivity was made by an image analyser. The level of neuronal nitric oxide synthase protein was measured by the Western blot analysis. 3. Our data show the increase of inducible nitric oxide synthase activity and a decrease of Ca(2+)-dependent nitric oxide synthase activity in the injured site analysed 1 and 7 days after surgery. In segments remote from the epicentre of injury the inducible nitric oxide synthase activity was increased at both time points. Ca(2+)-dependent nitric oxide synthase activity had decreased in L5-S1 segments in a group of animals surviving for 7 days. A hemisection performed at thoracic level did not cause significant difference in the nitric oxide synthase activities and in the level of neuronal nitric oxide synthase protein between the contra- and ipsilateral sides in C6-Th1 and L5-S1 segments taken as a whole. Significant differences were observed, but only when the spinal cord was analysed segment by segment, and/or was divided into dorsal and ventral parts. The cell counts in the cervicothoracic (C7-Th1) and lumbosacral (L5-S1) enlargements revealed changes in neuronal nitric oxide synthase immunoreactivity on the ipsilateral side of the injury. The densitometric area measurements confirmed the reduction of somal, neuropil and axonal neuronal nitric oxide synthase immunoreactive staining in the ventral part of rostrally oriented segments. 4. Our findings provide evidence that the changes in nitric oxide synthase pools are limited not only to impact zone, but spread outside the original lesion. The regional distribution of nitric oxide synthase activity and neuronal nitric oxide synthase immunoreactivity, measured segment by segment shows that nitric oxide may play a significant role in the stepping cycle in the quadrupeds.

1. The effect of hemisection of the cervical spinal cord on NADPH-diaphorase staining in the reticular nuclei of the rabbit medulla was investigated using histochemical technique. 2. A quantitative assessment of somal and neuropil NADPH-diaphorase staining was made by an image analyzer in a selected area of each reticular nucleus of the rabbit medulla. 3. On the 7th postsurgery day, the highest up-regulation of somatic NADPH-diapho- rase staining was observed in regions regulating cardiorespiratory processes; however, the highest increase of neuropil NADPH-diaphorase staining was found in the reticular nuclei modulating the tonus of postural muscles. 4. The degeneration of non-NADPH-diaphorase-stained neurons was detected throughout the reticular formation of the medulla, but the extent of neuronal death did not correlate with the up-regulation of the NADPH-diaphorase staining in the reticular nuclei of the medulla. 5. The findings provide evidence that NADPH-diaphorase-exhibiting neurons are refractory to the hemisection of the cervical spinal cord and that the neuronal up-regulation of NADPH-diaphorase at the medullar level is probably not a causative factor leading to the death of the reticulospinal neurons.

In this study we investigate the occurrence and origin of punctate nitric oxide synthase immunoreactivity in the neuropil of the ventral motor nucleus in C7-Th1 segments of the dog spine, which are supposed to be the terminal field of an ascending premotor propriospinal nitric oxide synthase-immunoreactive pathway. As the first step, nitric oxide synthase immunohistochemistry was used to distinguish nitric oxide synthase-immunoreactive staining of the ventral motor nucleus. Dense, punctate nitric oxide synthase immunoreactivity was found on control sections in the neuropil of the ventral motor nucleus. After hemisection at Th10–11, axotomy-induced retrograde changes consisting in a strong upregulation of nitric oxide synthase-containing neurons were found mostly unilaterally in lamina VIII, the medial part of lamina VII and in the pericentral region in all segments of the lumbosacral enlargement. Concurrently, a strong depletion of the punctate nitric oxide synthase immunopositivity in the neuropil of the ventral motor nucleus ipsilaterally with the hemisection was detected, thus revealing that an uncrossed ascending premotor propriospinal pathway containing a fairly high number of nitric oxide synthase-immunoreactive fibers terminates in the ventral motor nucleus. Application of the retrograde fluorescent tracer Fluorogold injected into the ventral motor nucleus and analysis of alternate sections processed for nitric oxide synthase immunocytochemistry revealed the presence of Fluorogold-labeled and nitric oxide synthase-immunoreactive axons in the ventrolateral funiculus and in the lateral and medial portions of the ventral column throughout the thoracic and upper lumbar segments. A noticeable number of Fluorogold-labeled and nitric oxide synthase-immunoreactive somata detected on consecutive sections were found in the lumbosacral enlargement, mainly in laminae VIII–IX, the medial part of lamina VII and in the pericentral region (lamina X), ipsilaterally with the injection of Fluorogold into the ventral motor nucleus. In summary, the present study provides evidence for a hitherto unknown ascending premotor propriospinal nitric oxide synthase-immunoreactive pathway connecting the lumbosacral enlargement with the motoneurons of the ventral motor nucleus in the dog.

Obesity is a major health problem worldwide. We are studying the causes and effects of obesity in C57Bl/6 mice following genetic ablation of NG2, a chondroitin sulfate proteoglycan widely expressed in progenitor cells and also in adipocytes. Although global NG2 ablation delays early postnatal adipogenesis in mouse skin, adult NG2 null mice are paradoxically heavier than wild-type mice, exhibiting larger white fat deposits. This adult onset obesity is not due to NG2-dependent effects on CNS function, since specific ablation of NG2 in oligodendrocyte progenitors yields the opposite phenotype; i.e. abnormally lean mice. Metabolic analysis reveals that, while activity and food intake are unchanged in global NG2 null mice, O(2) consumption and CO(2) production are decreased, suggesting a decrease in energy expenditure. Since brown fat plays important roles in regulating energy expenditure, we have investigated brown fat function via cold challenge and high fat diet feeding, both of which induce the adaptive thermogenesis that normally occurs in brown fat. In both tests, body temperatures in NG2 null mice are reduced compared to wild-type mice, indicating a deficit in brown fat function in the absence of NG2. In addition, adipogenesis in NG2 null brown pre-adipocytes is dramatically impaired compared to wild-type counterparts. Moreover, mRNA levels for PR domain containing 16 (PRDM16) and peroxisome proliferator-activated receptor γ coactivator (PGC)1-α, proteins important for brown adipocyte differentiation, are decreased in NG2 null brown fat deposits in vivo and NG2 null brown pre-adipocytes in vitro. Altogether, these results indicate that brown fat dysfunction in NG2 null mice results from deficits in the recruitment and/or development of brown pre-adipocytes. As a consequence, obesity in NG2 null mice may occur due to disruptions in brown fat-dependent energy homeostasis, with resulting effects on lipid storage in white adipocytes.

Histochemical, immunocytochemical and radioassay study was performed to detect the occurrence of NOS-immunoreactive primary trigeminal sensory somata in the trigeminal ganglion, including their fiber components. Spinal trigeminal tract and sensory trigeminal nuclei were studied using the same methods. It was found that more than 30% of all somata in the trigeminal ganglion are NOS immunoreactive. Corresponding fibers were detected in the spinal trigeminal tract. NOS immunoreactive fibers of three different categories could be followed to terminate in the sensory trigeminal nuclei. Data presented here confirm that trigeminal sensory system is richly endowed with NOS and that NO is used to communicate between the first and second-order trigeminal sensory neurons. Acknowledgements: Supported by VEGA Grant no. 2/3217/23PS9, STAA Grant no. 51-013002 and by NIH grants NS 32794 and NS 40386 to M.M.

Background The NG2 proteoglycan is expressed by several cell types in demyelinated lesions and has important effects on the biology of these cells. Here we determine the cell-type-specific roles of NG2 in the oligodendrocyte progenitor cell (OPC) and myeloid cell contributions to demyelination and remyelination. Methods We have used Cre-Lox technology to dissect the cell-type-specific contributions of NG2 to myelin damage and repair. Demyelination is induced by microinjection of 1 % lysolecithin into the spinal cord white matter of control, OPC-specific NG2-null (OPC-NG2ko), and myeloid-specific NG2-null (My-NG2ko) mice. The status of OPCs, myeloid cells, axons, and myelin is assessed by light, immunofluorescence, confocal, and electron microscopy. Results In OPC-NG2ko mice 1 week after lysolecithin injection, the OPC mitotic index is reduced by 40 %, resulting in 25 % fewer OPCs at 1 week and a 28 % decrease in mature oligodendrocytes at 6 weeks post-injury. The initial demyelinated lesion size is not affected in OPC-NG2ko mice, but lesion repair is delayed by reduced production of oligodendrocytes. In contrast, both the initial extent of demyelination and the kinetics of lesion repair are decreased in My-NG2ko mice. Surprisingly, the OPC mitotic index at 1 week post-injury is also reduced (by 48 %) in My-NG2ko mice, leading to a 35 % decrease in OPCs at 1 week and a subsequent 34 % reduction in mature oligodendrocytes at 6 weeks post-injury. Clearance of myelin debris is also reduced by 40 % in My-NG2ko mice. Deficits in myelination detected by immunostaining for myelin basic protein are confirmed by toluidine blue staining and by electron microscopy. In addition to reduced myelin repair, fewer axons are found in 6-week lesions in both OPC-NG2ko and My-NG2ko mice, emphasizing the importance of myelination for neuron survival. Conclusions Reduced generation of OPCs and oligodendrocytes in OPC-NG2ko mice correlates with reduced myelin repair. Diminished demyelination in My-NG2ko mice may stem from a reduction (approximately 70 %) in myeloid cell recruitment to lesions. Reduced macrophage/microglia numbers may then result in decreased myelin repair via diminished clearance of myelin debris and reduced stimulatory effects on OPCs.