Integration and Long Distance Axonal Regeneration in the Central Nervous System from Transplanted Primitive Neural Stem Cells

Spinal cord injury (SCI) results in devastating motor and sensory deficits secondary to disrupted neuronal circuits and poor regenerative potential. Efforts to promote regeneration through cell extrinsic and intrinsic manipulations have met with limited success. Stem cells represent an as yet unrealized therapy in SCI. Recently, we identified novel culture methods to induce and maintain primitive neural stem cells (pNSCs) from human embryonic stem cells. We tested whether transplanted human pNSCs can integrate into the CNS of the developing chick neural tube and injured adult rat spinal cord. Following injection of pNSCs into the developing chick CNS, pNSCs integrated into the dorsal aspects of the neural tube, forming cell clusters that spontaneously differentiated into neurons. Furthermore, following transplantation of pNSCs into the lesioned rat spinal cord, grafted pNSCs survived, differentiated into neurons, and extended long distance axons through the scar tissue at the graft-host interface and into the host spinal cord to form terminal-like structures near host spinal neurons. Together, these findings suggest that pNSCs derived from human embryonic stem cells differentiate into neuronal cell types with the potential to extend axons that associate with circuits of the CNS and, more importantly, provide new insights into CNS integration and axonal regeneration, offering hope for repair in SCI.     

Small-molecule-induced Rho-inhibition: NSAIDs after spinal cord injury

Limited axonal plasticity within the central nervous system (CNS) is a major restriction for functional recovery after CNS injury. The small GTPase RhoA is a key molecule of the converging downstream cascade that leads to the inhibition of axonal re-growth. The Rho-pathway integrates growth inhibitory signals derived from extracellular cues, such as chondroitin sulfate proteoglycans, Nogo-A, myelin-associated glycoprotein, oligodendrocyte-myelin glycoprotein, Ephrins and repulsive guidance molecule-A, into the damaged axon. Consequently, the activation of RhoA results in growth cone collapse and finally outgrowth failure. In turn, the inhibition of RhoA-activation blinds the injured axon to its growth inhibitory environment resulting in enhanced axonal sprouting and plasticity. This has been demonstrated in various CNS-injury models for direct RhoA-inhibition and for downstream/upstream blockade of the RhoA-associated pathway. In addition, RhoA-inhibition reduces apoptotic cell death and secondary damage and improves locomotor recovery in clinically relevant models after experimental spinal cord injury (SCI). Unexpectedly, a subset of "small molecules" from the group of non-steroid anti-inflammatory drugs, particularly the FDA-approved ibuprofen, has recently been identified as (1) inhibiting RhoA-activation, (2) enhancing axonal sprouting/regeneration, (3) protecting "tissue at risk" (neuroprotection) and (4) improving motor recovery confined to realistic therapeutical time-frames in clinically relevant SCI models. Here, we survey the effect of small-molecule-induced RhoA-inhibition on axonal plasticity and neurofunctional outcome in CNS injury paradigms. Furthermore, we discuss the body of preclinical evidence for a possible clinical translation with a focus on ibuprofen and illustrate putative risks and benefits for the treatment of acute SCI.     

Locomotor Dysfunction and Pain: The Scylla and Charybdis of Fiber Sprouting After Spinal Cord Injury

Injury to the spinal cord (SCI) can produce a constellation of problems including chronic pain, autonomic dysreflexia, and motor dysfunction. Neuroplasticity in the form of fiber sprouting or the lack thereof is an important phenomenon that can contribute to the deleterious effects of SCI. Aberrant sprouting of primary afferent fibers and synaptogenesis within incorrect dorsal horn laminae leads to the development and maintenance of chronic pain as well as autonomic dysreflexia. At the same time, interruption of connections between supraspinal motor control centers and spinal cord output cells, due to lack of successful regenerative sprouting of injured descending fiber tracts, contributes to motor deficits. Similarities in the molecular control of axonal growth of motor and sensory fibers have made the development of cogent therapies difficult. In this study, we discuss recent findings related to the degradation of inhibitory barriers and promotion of sprouting of motor fibers as a strategy for the restoration of motor function and note that this may induce primary afferent fiber sprouting that can contribute to chronic pain. We highlight the importance of careful attentiveness to off-target molecular- and circuit-level modulation of nociceptive processing while moving forward with the development of therapies that will restore motor function after SCI.     

Achieving CNS axon regeneration by manipulating convergent neuro-immune signaling

After central nervous system (CNS) trauma, axons have a low capacity for regeneration. Regeneration failure is associated with a muted regenerative response of the neuron itself, combined with a growth-inhibitory and cytotoxic post-injury environment. After spinal cord injury (SCI), resident and infiltrating immune cells (especially microglia/macrophages) contribute significantly to the growth-refractory milieu near the lesion. By targeting both the regenerative potential of the axon and the cytotoxic phenotype of microglia/macrophages, we may be able to improve CNS repair after SCI. In this review, we discuss molecules shown to impact CNS repair by affecting both immune cells and neurons. Specifically, we provide examples of pattern recognition receptors, integrins, cytokines/chemokines, nuclear receptors and galectins that could improve CNS repair. In many cases, signaling by these molecules is complex and may have contradictory effects on recovery depending on the cell types involved or the model studied. Despite this caveat, deciphering convergent signaling pathways on immune cells (which affect axon growth indirectly) and neurons (direct effects on axon growth) could improve repair and recovery after SCI. Future studies must continue to consider how regenerative therapies targeting neurons impact other cells in the pathological CNS. By identifying molecules that simultaneously improve axon regenerative capacity and drive the protective, growth-promoting phenotype of immune cells, we may discover SCI therapies that act synergistically to improve CNS repair and functional recovery.     

The role of repulsive guidance molecules in the embryonic and adult vertebrate central nervous system

During the development of the nervous system, outgrowing axons often have to travel long distances to reach their target neurons. In this process, outgrowing neurites tipped with motile growth cones rely on guidance cues present in their local environment. These cues are detected by specific receptors expressed on growth cones and neurites and influence the trajectory of the growing fibres. Neurite growth, guidance, target innervation and synapse formation and maturation are the processes that occur predominantly but not exclusively during embryonic or early post-natal development in vertebrates. As a result, a functional neural network is established, which is usually remarkably stable. However, the stability of the neural network in higher vertebrates comes at an expensive price, i.e. the loss of any significant ability to regenerate injured or damaged neuronal connections in their central nervous system (CNS). Most importantly, neurite growth inhibitors prevent any regenerative growth of injured nerve fibres. Some of these inhibitors are associated with CNS myelin, others are found at the lesion site and in the scar tissue. Traumatic injuries in brain and spinal cord of mammals induce upregulation of embryonic inhibitory or repulsive guidance cues and their receptors on the neurites. An example for embryonic repulsive directional cues re-expressed at lesion sites in both the rat and human CNS is provided with repulsive guidance molecules, a new family of directional guidance cues.     

The role of mTOR signaling pathway in spinal cord injury

The mammalian target of rapamycin (mTOR) signaling pathway plays an important role in multiple cellular functions, such as cell metabolism, proliferation and survival. Many previous studies have shown that mTOR regulates both neuroprotective and neuroregenerative functions in trauma and various diseases in the central nervous system (CNS). Recently, we reported that inhibition of mTOR using rapamycin reduces neural tissue damage and locomotor impairment after spinal cord injury (SCI) in mice. Our results demonstrated that the administration of rapamycin at four hours after injury significantly increases the activity of autophagy and reduces neuronal loss and cell death in the injured spinal cord. Furthermore, rapamycin-treated mice show significantly better locomotor function in the hindlimbs following SCI than vehicle-treated mice. These findings indicate that the inhibition of mTOR signaling using rapamycin during the acute phase of SCI produces neuroprotective effects and reduces secondary damage at lesion sites. However, the role of mTOR signaling in injured spinal cords has not yet been fully elucidated. Various functions are regulated by mTOR signaling in the CNS, and multiple pathophysiological processes occur following SCI. Here, we discuss several unresolved issues and review the evidence from related articles regarding the role and mechanisms of the mTOR signaling pathway in neuroprotection and neuroregeneration after SCI.     

The role of mTOR signaling pathway in spinal cord injury

The mammalian target of rapamycin (mTOR) signaling pathway plays an important role in multiple cellular functions, such as cell metabolism, proliferation and survival. Many previous studies have shown that mTOR regulates both neuroprotective and neuroregenerative functions in trauma and various diseases in the central nervous system (CNS). Recently, we reported that inhibition of mTOR using rapamycin reduces neural tissue damage and locomotor impairment after spinal cord injury (SCI) in mice. Our results demonstrated that the administration of rapamycin at four hours after injury significantly increases the activity of autophagy and reduces neuronal loss and cell death in the injured spinal cord. Furthermore, rapamycin-treated mice show significantly better locomotor function in the hindlimbs following SCI than vehicle-treated mice. These findings indicate that the inhibition of mTOR signaling using rapamycin during the acute phase of SCI produces neuroprotective effects and reduces secondary damage at lesion sites. However, the role of mTOR signaling in injured spinal cords has not yet been fully elucidated. Various functions are regulated by mTOR signaling in the CNS, and multiple pathophysiological processes occur following SCI. Here, we discuss several unresolved issues and review the evidence from related articles regarding the role and mechanisms of the mTOR signaling pathway in neuroprotection and neuroregeneration after SCI.     

Regeneration of sensory axons within the injured spinal cord induced by intraganglionic cAMP elevation

The peripheral branch of primary sensory neurons regenerates after injury, but there is no regeneration when their central branch is severed by spinal cord injury. Here we show that microinjection of a membrane-permeable analog of cAMP in lumbar dorsal root ganglia markedly increases the regeneration of injured central sensory branches. The injured axons regrow into the spinal cord lesion, often traversing the injury site. This result mimics the effect of a conditioning peripheral nerve lesion. We also demonstrate that sensory neurons exposed to cAMP in vivo, when subsequently cultured in vitro, show enhanced growth of neurites and an ability to overcome inhibition by CNS myelin. Thus, stimulating cAMP signaling increases the intrinsic growth capacity of injured sensory axons. This approach may be useful in promoting regeneration after spinal cord injury.     

Alterations in the expression of the apurinic/apyrimidinic endonuclease-1/redox factor-1 (APE/ref-1) and DNA damage in the caudal region of acute and chronic spinal cord injured rats treated by embryonic neural stem cells

The oxidative mechanisms of injury-induced damage of neurons within the spinal cord are not very well understood. We used a model of T8-T9 spinal cord injury (SCI) in the rat to induce neuronal degeneration. In this spinal cord injury model, unilateral avulsion of the spinal cord causes oxidative stress of neurons. We tested the hypothesis that apurinic/apyrimidinic endonuclease (or redox effector factor-1, APE/Ref-1) regulates this neuronal oxidation mechanism in the spinal cord region caudal to the lesion, and that DNA damage is an early upstream signal. The embryonic neural stem cell therapy significantly decreased DNA-damage levels in both study groups - acutely (followed up to 7 days after SCI), and chronically (followed up to 28 days after SCI) injured animals. Meanwhile, mRNA levels of APE/Ref-1 significantly increased after embryonic neural stem cell therapy in acutely and chronically injured animals when compared to acute and chronic sham groups. Our data has demonstrated that an increase of APE/Ref-1 mRNA levels in the caudal region of spinal cord strongly correlated with DNA damage after traumatic spinal cord injury. We suggest that DNA damage can be observed both in lesional and caudal regions of the acutely and chronically injured groups, but DNA damage is reduced with embryonic neural stem cell therapy.     

The Nogo receptor, its ligands and axonal regeneration in the spinal cord; A review

At least three proteins present in CNS myelin, Nogo, MAG and OMgp are capable of causing growth cone collapse and inhibiting neurite outgrowth in vitro. Surprisingly, Nogo and OMgp are also strongly expressed by many neurons (including neocortical projection cells). Nogo expression is increased by some cells at the borders of CNS lesion sites and by cells in injured peripheral nerves, but Nogo and CNS myelin are largely absent from spinal cord injury sites, which are none the less strongly inhibitory to axonal regeneration. Nogo is found on growing axons during development, suggesting possible functions for neuronal Nogo in axon guidance. Although Nogo, MAG and OMgp lack sequence homologies, they all bind to the Nogo receptor (NgR), a GPI-linked cell surface molecule which, in turn, binds p75 to activate RhoA. NgR is strongly expressed by cerebral cortical neurons but many other neurons express NgR weakly or not at all. Some neurons, such as DRG cells, respond to Nogo and CNS myelin in vitro although they express little or no NgR in vivo which, with other data, indicates that other receptors are available for NgR ligands. NgR expression is unaffected by injury to the nervous system, and there is no clear correlation between NgR expression by neurons and lack of regenerative ability. In the injured spinal cord, interactions between NgR and its ligands are most likely to be important for limiting regeneration of corticospinal and some other descending tracts; other receptors may be more important for ascending tracts. Antibodies to Nogo, mainly the poorly-characterised IN-1 or its derivatives, have been shown to enhance recovery from partial transections of the spinal cord. They induce considerable plasticity from the axons of corticospinal neurons, including sprouting across the midline and, to a limited extent, regeneration around the lesion. Regeneration of corticospinal axons induced by Nogo antibodies has not yet been demonstrated after complete transections or contusion injuries of the spinal cord. It is not clear whether antibodies against Nogo act on oligodendrocytes/myelin or by binding to neuronal Nogo, or whether they can stimulate regeneration of ascending axons in the spinal cord, most of which express little or no NgR. Despite these uncertainties, however, NgR and its ligands offer important new targets for enhancing plasticity and regeneration in the nervous system.     

p53- and Bax-Mediated Apoptosis in Injured Rat Spinal Cord

Spinal cord injury (SCI) induces a series of endogenous biochemical changes that lead to secondary degeneration, including apoptosis. p53-mediated mitochondrial apoptosis is likely to be an important mechanism of cell death in spinal cord injury. However, the signaling cascades that are activated before DNA fragmentation have not yet been determined. DNA damage-induced, p53-activated neuronal cell death has already been identified in several neurodegenerative diseases. To determine DNA damage-induced, p53-mediated apoptosis in spinal cord injury, we performed RT-PCR microarray and analyzed 84 DNA damaging and apoptotic genes. Genes involved in DNA damage and apoptosis were upregulated whereas anti-apoptotic genes were downregulated in injured spinal cords. Western blot analysis showed the upregulation of DNA damage-inducing protein such as ATM, cell cycle checkpoint kinases, 8-hydroxy-2′-deoxyguanosine (8-OHdG), BRCA2 and H2AX in injured spinal cord tissues. Detection of phospho-H2AX in the nucleus and release of 8-OHdG in cytosol were demonstrated by immunohistochemistry. Expression of p53 was observed in the neurons, oligodendrocytes and astrocytes after spinal cord injury. Upregulation of phospho-p53, Bax and downregulation of Bcl2 were detected after spinal cord injury. Sub-cellular distribution of Bax and cytochrome c indicated mitochondrial-mediated apoptosis taking place after spinal cord injury. In addition, we carried out immunohistochemical analysis to confirm Bax translocation into the mitochondria and activated p53 at Ser³⁹². Expression of APAF1, caspase 9 and caspase 3 activities confirmed the intrinsic apoptotic pathway after SCI. Activated p53 and Bax mitochondrial translocation were detected in injured spinal neurons. Taken together, the in vitro data strengthened the in vivo observations of DNA damage-induced p53-mediated mitochondrial apoptosis in the injured spinal cord.     

Spinal cord injury triggers systemic autoimmunity: evidence for chronic B lymphocyte activation and lupus-like autoantibody synthesis

Clinical and experimental data indicate that spinal cord injury (SCI) elicits pathological T-cell responses. Implicit in these data, but poorly understood, is that B lymphocytes (B cells) also contribute to the delayed pathophysiology of spinal trauma. Here, for the first time, we show that experimental spinal contusion injury elicits chronic systemic and intraspinal B cell activation with the emergence of a B cell-dependent organ-specific and systemic autoimmune response. Specifically, using sera from spinal cord injured mice, immunoblots reveal oligoclonal IgG reactivity against multiple CNS proteins. We also show SCI-induced synthesis of autoantibodies that bind nuclear antigens including DNA and RNA. Elevated levels of anti-DNA antibodies are a distinguishing feature of systemic lupus erythematosus and, via their ability to cross-react with neuronal antigens, can cause neuropathology. We show a similar pathologic potential for the autoantibodies produced after SCI. Thus, mammalian SCI produces marked dysregulation of B cell function (i.e. autoimmunity) with pathological potential.     

Nerve injury signaling

Although neurons within the peripheral nervous system (PNS) have a remarkable ability to repair themselves after injury, neurons within the central nervous system (CNS) do not spontaneously regenerate. This problem has remained recalcitrant despite a century of research on the reaction of axons to injury. The balance between inhibitory cues present in the environment and the intrinsic growth capacity of the injured neuron determines the extent of axonal regeneration following injury. The cell body of an injured neuron must receive accurate and timely information about the site and extent of axonal damage in order to increase its intrinsic growth capacity and successfully regenerate. One of the mechanisms contributing to this process is retrograde transport of injury signals. For example, molecules activated at the injury site convey information to the cell body leading to the expression of regeneration-associated genes and increased growth capacity of the neuron. Here we discuss recent studies that have begun to dissect the injury-signaling pathways involved in stimulating the intrinsic growth capacity of injured neurons.     

A selective Sema3A inhibitor enhances regenerative responses and functional recovery of the injured spinal cord

Axons in the adult mammalian central nervous system (CNS) exhibit little regeneration after injury. It has been suggested that several axonal growth inhibitors prevent CNS axonal regeneration. Recent research has demonstrated that semaphorin3A (Sema3A) is one of the major inhibitors of axonal regeneration. We identified a strong and selective inhibitor of Sema3A, SM-216289, from the fermentation broth of a fungal strain. To examine the effect of SM-216289 in vivo, we transected the spinal cord of adult rats and administered SM-216289 into the lesion site for 4 weeks. Rats treated with SM-216289 showed substantially enhanced regeneration and/or preservation of injured axons, robust Schwann cell-mediated myelination and axonal regeneration in the lesion site, appreciable decreases in apoptotic cell number and marked enhancement of angiogenesis, resulting in considerably better functional recovery. Thus, Sema3A is essential for the inhibition of axonal regeneration and other regenerative responses after spinal cord injury (SCI). These results support the possibility of using Sema3A inhibitors in the treatment of human SCI.     

Rho activation patterns after spinal cord injury and the role of activated Rho in apoptosis in the central nervous system

Growth inhibitory proteins in the central nervous system (CNS) block axon growth and regeneration by signaling to Rho, an intracellular GTPase. It is not known how CNS trauma affects the expression and activation of RhoA. Here we detect GTP-bound RhoA in spinal cord homogenates and report that spinal cord injury (SCI) in both rats and mice activates RhoA over 10-fold in the absence of changes in RhoA expression. In situ Rho-GTP detection revealed that both neurons and glial cells showed Rho activation at SCI lesion sites. Application of a Rho antagonist (C3-05) reversed Rho activation and reduced the number of TUNEL-labeled cells by approximately 50% in both injured mouse and rat, showing a role for activated Rho in cell death after CNS injury. Next, we examined the role of the p75 neurotrophin receptor (p75NTR) in Rho signaling. After SCI, an up-regulation of p75NTR was detected by Western blot and observed in both neurons and glia. Treatment with C3-05 blocked the increase in p75NTR expression. Experiments with p75NTR-null mutant mice showed that immediate Rho activation after SCI is p75NTR dependent. Our results indicate that blocking overactivation of Rho after SCI protects cells from p75NTR-dependent apoptosis.     

Neurotrophic factors in combinatorial approaches for spinal cord regeneration

Axonal regeneration is inhibited by a plethora of different mechanisms in the adult central nervous system (CNS). While neurotrophic factors have been shown to stimulate axonal growth in numerous animal models of nervous system injury, a lack of suitable growth substrates, an insufficient activation of neuron-intrinsic regenerative programs, and extracellular inhibitors of regeneration limit the efficacy of neurotrophic factor delivery for anatomical and functional recovery after spinal cord injury. Thus, growth-stimulating factors will likely have to be combined with other treatment approaches to tap into the full potential of growth factor therapy for axonal regeneration. In addition, the temporal and spatial distribution of growth factors have to be tightly controlled to achieve biologically active concentrations, to allow for the chemotropic guidance of axons, and to prevent adverse effects related to the widespread distribution of neurotrophic factors. Here, we will review the rationale for combinatorial treatments in axonal regeneration and summarize some recent progress in promoting axonal regeneration in the injured CNS using such approaches.     

Effects of combinatorial treatment with pituitary adenylate cyclase activating peptide and human mesenchymal stem cells on spinal cord tissue repair

The aim of this study is to understand if human mesenchymal stem cells (hMSCs) and neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP) have synergistic protective effect that promotes functional recovery in rats with severe spinal cord injury (SCI). To evaluate the effect of delayed combinatorial therapy of PACAP and hMSCs on spinal cord tissue repair, we used the immortalized hMSCs that retain their potential of neuronal differentiation under the stimulation of neurogenic factors and possess the properties for the production of several growth factors beneficial for neural cell survival. The results indicated that delayed treatment with PACAP and hMSCs at day 7 post SCI increased the remaining neuronal fibers in the injured spinal cord, leading to better locomotor functional recovery in SCI rats when compared to treatment only with PACAP or hMSCs. Western blotting also showed that the levels of antioxidant enzymes, Mn-superoxide dismutase (MnSOD) and peroxiredoxin-1/6 (Prx-1 and Prx-6), were increased at the lesion center 1 week after the delayed treatment with the combinatorial therapy when compared to that observed in the vehicle-treated control. Furthermore, in vitro studies showed that co-culture with hMSCs in the presence of PACAP not only increased a subpopulation of microglia expressing galectin-3, but also enhanced the ability of astrocytes to uptake extracellular glutamate. In summary, our in vivo and in vitro studies reveal that delayed transplantation of hMSCs combined with PACAP provides trophic molecules to promote neuronal cell survival, which also foster beneficial microenvironment for endogenous glia to increase their neuroprotective effect on the repair of injured spinal cord tissue.     

Chemokines in CNS injury and repair

Recruitment of inflammatory cells is known to drive the secondary damage cascades that are common to injuries of the central nervous system (CNS). Cell activation and infiltration to the injury site is orchestrated by changes in the expression of chemokines, the chemoattractive cytokines. Reducing the numbers of recruited inflammatory cells by the blocking of the action of chemokines has turned out be a promising approach to diminish neuroinflammation and to improve tissue preservation and neovascularization. In addition, several chemokines have been shown to be essential for stem/progenitor cell attraction, their survival, differentiation and cytokine production. Thus, chemokines might indirectly participate in remyelination, neovascularization and neuroprotection, which are important prerequisites for CNS repair after trauma. Moreover, CXCL12 promotes neurite outgrowth in the presence of growth inhibitory CNS myelin and enhances axonal sprouting after spinal cord injury (SCI). Here, we review current knowledge about the exciting functions of chemokines in CNS trauma, including SCI, traumatic brain injury and stroke. We identify common principles of chemokine action and discuss the potentials and challenges of therapeutic interventions with chemokines.     

Neuronal glycosylation differentials in normal, injured and chondroitinase-treated environments

Glycosylation is found ubiquitously throughout the central nervous system (CNS). Chondroitin sulphate proteoglycans (CSPGs) are a group of molecules heavily substituted with glycosaminoglycans (GAGs) and are found in the extracellular matrix (ECM) and cell surfaces. Upon CNS injury, a glial scar is formed, which is inhibitory for axon regeneration. Several CSPGs are up-regulated within the glial scar, including NG2, and these CSPGs are key inhibitory molecules of axonal regeneration. Treatment with chondroitinase ABC (ChABC) can neutralise the inhibitory nature of NG2. A gene expression dataset was mined in silico to verify differentially regulated glycosylation-related genes in neurons after spinal cord injury and identify potential targets for further investigation. To establish the glycosylation differential of neurons that grow in a healthy, inhibitory and ChABC-treated environment, we established an indirect co-culture system where PC12 neurons were grown with primary astrocytes, Neu7 astrocytes (which overexpress NG2) and Neu7 astrocytes treated with ChABC. After 1, 4 and 8 days culture, lectin cytochemistry of the neurons was performed using five fluorescently-labelled lectins (ECA MAA, PNA, SNA-I and WFA). Usually α-(2,6)-linked sialylation scarcely occurs in the CNS but this motif was observed on the neurons in the injured environment only at day 8. Treatment with ChABC was successful in returning neuronal glycosylation to normal conditions at all timepoints for MAA, PNA and SNA-I staining, and by day 8 in the case of WFA. This study demonstrated neuronal cell surface glycosylation changes in an inhibitory environment and indicated a return to normal glycosylation after treatment with ChABC, which may be promising for identifying potential therapies for neuronal regeneration strategies.