CNS Injury Models Research Articles

Role of IGF-1 in neuroinflammation and cognition deficits induced by sleep deprivation

Sleep deprivation negatively influences cognition, however, the regulatory mechanisms to counteract this effect have not been identified. IGF-1 has been shown to be anti-inflammatory and neuroprotective in CNS injury models. In this study, we determined the impact of IGF-1 on brain injury and inflammation while modeling sleep deprivation. We found that IGF-1 was downregulated in human peripheral blood and in mice subjected to sleep deprivation for 5 days, with reduced activation of the downstream PI3K/AKT/GSK-3β pathway in mice brains. In addition, we found reduced levels of the anti-apoptosis enzyme Bcl-2 and increased levels of pro-apoptosis enzyme Caspase-9 expression, together with increased pro-inflammatory factors. The administration of IGF-1 after sleep deprivation induced activation of the PI3K/AKT/GSK-3β pathway, reversed changes in Bcl-2, Caspase-9, and pro-inflammatory factors, and alleviated cognitive impairment. Notably, IGF-1 also induced activation of the PI3K/AKT/GSK-3β pathway, and displayed anti-apoptosis and anti-inflammatory properties under normal sleep conditions,while IGF-1 did not improve the cognition under normal sleep conditions. These results suggest that the IGF-1/PI3K/AKT/GSK-3β pathway is involved in the regulation of cognitive function after sleep deprivation through modulation of apoptosis and inflammatory response. IGF-1 could be a viable therapeutic target, though further investigation is required to better understand its role in sleep deprivation.

Repetitive Mild Closed Head Injury in Adolescent Mice Is Associated with Impaired Proteostasis, Neuroinflammation, and Tauopathy.

Repetitive mild traumatic brain injury (mTBI) in children and adolescents leads to acute and chronic neurologic sequelae and is linked to later life neurodegenerative disease. However, the biological mechanisms connecting early life mTBI to neurodegeneration remain unknown. Using an adolescent mouse repetitive closed head injury model that induces progressive cognitive impairment in males and anxiety in females in the absence of overt histopathology, we examined transcriptional and translational changes in neurons isolated from sham and injured brain in the chronic phase after injury. At 14 months, single-nuclei RNA sequencing of cortical brain tissue identified disruption of genes associated with neuronal proteostasis and evidence for disrupted ligand-receptor signaling networks in injured mice. Western blot analysis of isolated neurons showed evidence of inflammasome activation and downstream IL-1β processing, as previously demonstrated in acute CNS injury models, and accumulation of misfolded, hyperphosphorylated tau, and changes in expression of proteins suggestive of impaired translation in males but not in females. At 6 months, injured IL-1 receptor 1 (IL-1R1) KO mice, which are protected from postinjury cognitive deficits, had decreased accumulation of pro-IL-1β and misfolded tau in cortex and cerebellum, suggesting that IL-1R1 is upstream of inflammasome priming (defined as increase in pro-IL-1β) and abnormal tau phosphorylation. Together, our findings provide evidence for neuronal inflammasome activation and impaired proteostasis as key mechanisms linking repetitive mTBI in adolescence to later life neurologic dysfunction and neurodegeneration.SIGNIFICANCE STATEMENT Repetitive mild closed head injury in adolescent male mice leads to impaired proteostasis, tau phosphorylation, and inflammasome activation in neurons later in adulthood through mechanisms involving IL-1 receptor 1. The data are the first to link repetitive mild traumatic brain injury in adolescence to neurodegeneration and suggest molecular targets and pathways to prevent neurologic sequelae in the chronic period after injuries.

Effects of amyloid precursor protein peptide APP96-110, alone or with human mesenchymal stromal cells, on recovery after spinal cord injury.

Delivery of a peptide (APP96-110), derived from amyloid precursor protein (APP), has been shown to elicit neuroprotective effects following cerebral stroke and traumatic brain injury. In this study, the effect of APP96-110 or a mutant version of this peptide (mAPP96-110) was assessed following moderate (200 kdyn, (2 N)) thoracic contusive spinal cord injury (SCI) in adult Nude rats. Animals received a single tail vein injection of APP96-110 or mAPP96-110 at 30 minutes post-SCI and were then assessed for functional improvements over the next 8 weeks. A cohort of animals also received transplants of either viable or non-viable human mesenchymal stromal cells (hMSCs) into the SC lesion site at one week post-injury to assess the effect of combining intravenous APP96-110 delivery with hMSC treatment. Rats were perfused 8 weeks post-SCI and longitudinal sections of spinal cord analyzed for a number of factors including hMSC viability, cyst size, axonal regrowth, glial reactivity and macrophage activation. Analysis of sensorimotor function revealed occasional significant differences between groups using Ladderwalk or Ratwalk tests, however there were no consistent improvements in functional outcome after any of the treatments. mAPP96-110 alone, and APP96-110 in combination with both viable and non-viable hMSCs significantly reduced cyst size compared to SCI alone. Combined treatments with donor hMSCs also significantly increased βIII tubulin+, glial fibrillary acidic protein (GFAP+) and laminin+ expression, and decreased ED1+ expression in tissues. This preliminary study demonstrates that intravenous delivery of APP96-110 peptide has selective, modest neuroprotective effects following SCI, which may be enhanced when combined with hMSC transplantation. However, the effects are less pronounced and less consistent compared to the protective morphological and cognitive impact that this same peptide has on neuronal survival and behaviour after stroke and traumatic brain injury. Thus while the efficacy of a particular therapeutic approach in one CNS injury model may provide justification for its use in other neurotrauma models, similar outcomes may not necessarily occur and more targeted approaches suited to location and severity are required. All animal experiments were approved by The University of Western Australia Animal Ethics Committee (RA3/100/1460) on April 12, 2016.

Overexpression of Reticulon 3 Enhances CNS Axon Regeneration and Functional Recovery after Traumatic Injury.

CNS neurons are generally incapable of regenerating their axons after injury due to several intrinsic and extrinsic factors, including the presence of axon growth inhibitory molecules. One such potent inhibitor of CNS axon regeneration is Reticulon (RTN) 4 or Nogo-A. Here, we focused on RTN3 as its contribution to CNS axon regeneration is currently unknown. We found that RTN3 expression correlated with an axon regenerative phenotype in dorsal root ganglion neurons (DRGN) after injury to the dorsal columns, a well-characterised model of spinal cord injury. Overexpression of RTN3 promoted disinhibited DRGN neurite outgrowth in vitro and dorsal column axon regeneration/sprouting and electrophysiological, sensory and locomotor functional recovery after injury in vivo. Knockdown of protrudin, however, ablated RTN3-enhanced neurite outgrowth/axon regeneration in vitro and in vivo. Moreover, overexpression of RTN3 in a second model of CNS injury, the optic nerve crush injury model, enhanced retinal ganglion cell (RGC) survival, disinhibited neurite outgrowth in vitro and survival and axon regeneration in vivo, an effect that was also dependent on protrudin. These results demonstrate that RTN3 enhances neurite outgrowth/axon regeneration in a protrudin-dependent manner after both spinal cord and optic nerve injury.

Neuroprotective Effects of Curcumin-Loaded Emulsomes in a Laser Axotomy-Induced CNS Injury Model.

PurposeCurcumin, a polyphenol isolated from the rhizomes of turmeric, holds great potential as a neuroprotective agent in addition to its anti-inflammatory and antioxidant characteristics. The poor bioavailability and low stability of curcumin are the greatest barriers to its clinical use. This study aims to investigate the neuroprotective effect of curcumin on axonal injury, by delivering the lipophilic polyphenol to a primary hippocampal neuron culture by means of a lipid-based drug delivery system, named emulsomes.MethodsTo study neuroregeneration ex vivo, an injury model was established through single-cell laser axotomy on hippocampal neurites. Upon treatment with curcumin-loaded emulsomes (CurcuEmulsomes), curcumin and CurcuEmulsome uptake into neurons was verified by three-dimensional Z-stack images acquired with confocal microscopy. Neuron survival after axonal injury was tracked by propidium iodide (PI) and Hoechst staining. Alterations in expression levels of physiological markers, such as anti-apoptotic marker Bcl2, apoptotic marker cleaved caspase 3, neuroprotective marker Wnt3a and the neuronal survival marker mTOR, were investigated by immunocytochemistry analyses.ResultsThe results indicated significant improvement in the survival rate of injured neurons upon CurcuEmulsome treatment. Bcl2 expression was significantly higher for injured neurons treated with curcumin or CurcuEmulsome. Reduction in caspase 3 expression was seen in both curcumin and CurcuEmulsome treatment, whereas there were no significant changes in Wnt3a and mTOR expression.ConclusionThe established laser-axotomy model was proven as a reliable methodology to study neurodegenerative models ex vivo. CurcuEmulsomes delivered curcumin to primary hippocampal neurons successfully. Treated with CurcuEmulsomes, injured hippocampal neurons benefit from the neuroprotective effects of curcumin, exhibiting a higher survival rate and increased anti-apoptotic marker levels.

Intra-Arterial Delivery of Neural Stem Cells to the Rat and Mouse Brain: Application to Cerebral Ischemia.

Neural stem cell (NSC) therapy is an emerging innovative treatment for stroke, traumatic brain injury and neurodegenerative disorders. As compared to intracranial delivery, intra-arterial administration of NSCs is less invasive and produces a more diffuse distribution of NSCs within the brain parenchyma. Further, intra-arterial delivery allows the first-pass effect in the brain circulation, lessening the potential for trapping of cells in peripheral organs, such as liver and spleen, a complication associated with peripheral injections. Here, we detail the methodology, in both mice and rats, for delivery of NSCs through the common carotid artery (mouse) or external carotid artery (rat) to the ipsilateral hemisphere after an ischemic stroke. Using GFP-labeled NSCs, we illustrate the widespread distribution achieved throughout the rodent ipsilateral hemisphere at 1 d, 1 week and 4 weeks after postischemic delivery, with a higher density in or near the ischemic injury site. In addition to long-term survival, we show evidence of differentiation of GFP-labeled cells at 4 weeks. The intra-arterial delivery approach described here for NSCs can also be used for administration of therapeutic compounds, and thus has broad applicability to varied CNS injury and disease models across multiple species.

Acute Cellular and Functional Changes With a Combinatorial Treatment of Ion Channel Inhibitors Following Spinal Cord Injury.

Reducing the extent of secondary degeneration following spinal cord injury (SCI) is necessary to preserve function, but treatment options have thus far been limited. A combination of the ion channel inhibitors Lomerizine (Lom), YM872 and oxATP, to inhibit voltage-gated Ca2+ channels, Ca2+ permeable AMPA receptors, and purinergic P2X7 receptors respectively, effectively limits secondary consequences of injury in in vitro and in vivo models of CNS injury. Here, we investigated the efficacy of these inhibitors in a clinically relevant model of SCI. Fischer (F344) rats were subjected to a moderate (150 kD) contusive SCI at thoracic level T10 and assessed at 2 weeks or 10 weeks post-injury. Lom was delivered orally twice daily and YM872 and oxATP were delivered via osmotic mini-pump implanted at the time of SCI until 2 weeks following injury. Open field locomotion analysis revealed that treatment with the three inhibitors in combination improved the rate of functional recovery of the hind limb (compared to controls) as early as 1-day post-injury, with beneficial effects persisting to 14 days post-injury, while all three inhibitors were present. At 2 weeks following combinatorial treatment, the functional improvement was associated with significantly decreased cyst size, increased immunoreactivity of β-III tubulin+ve axons, myelin basic protein, and reduced lipid peroxidation by-products, and increased CC1+ve oligodendrocytes and NG2+ve/PDGFα+ve oligodendrocyte progenitor cell densities, compared to vehicle-treated SCI animals. The combination of Lom, oxATP, and YM872 shows preclinical promise for control of secondary degeneration following SCI, and further investigation of long-term sustained treatment is warranted.

Iron chelators inhibit amyloid-β-induced production of lipocalin 2 in cultured astrocytes

Lipocalin 2 (Lcn2) has been implicated to play a role in various neurodegenerative diseases, and normalizing its overexpression may be of therapeutic potential. Iron chelators were found to reduce Lcn2 levels in certain animal models of CNS injury. Focusing on Alzheimer's disease (AD), we found that the iron chelators deferoxamine and deferiprone inhibited amyloid-β (Aβ)-induced Lcn2 production in cultured primary astrocytes. Accordingly, Aβ-exposure increased astrocytic ferritin production, indicating the possibility that Aβ induces iron accumulation in astrocytes. This effect was not significantly modulated by Lcn2. Known neuroprotective effects of iron chelators may rely in part on normalization of Lcn2 levels.

Fractionated mitochondrial magnetic separation for isolation of synaptic mitochondria from brain tissue

While mitochondria maintain essential cellular functions, such as energy production, calcium homeostasis, and regulating programmed cellular death, they also play a major role in pathophysiology of many neurological disorders. Furthermore, several neurodegenerative diseases are closely linked with synaptic damage and synaptic mitochondrial dysfunction. Unfortunately, the ability to assess mitochondrial dysfunction and the efficacy of mitochondrial-targeted therapies in experimental models of neurodegenerative disease and CNS injury is limited by current mitochondrial isolation techniques. Density gradient ultracentrifugation (UC) is currently the only technique that can separate synaptic and non-synaptic mitochondrial sub-populations, though small brain regions cannot be assayed due to low mitochondrial yield. To address this limitation, we used fractionated mitochondrial magnetic separation (FMMS), employing magnetic anti-Tom22 antibodies, to develop a novel strategy for isolation of functional synaptic and non-synaptic mitochondria from mouse cortex and hippocampus without the usage of UC. We compared the yield and functionality of mitochondria derived using FMMS to those derived by UC. FMMS produced 3x more synaptic mitochondrial protein yield compared to UC from the same amount of tissue, a mouse hippocampus. FMMS also has increased sensitivity, compared to UC separation, to measure decreased mitochondrial respiration, demonstrated in a paradigm of mild closed head injury. Taken together, FMMS enables improved brain-derived mitochondrial yield for mitochondrial assessments and better detection of mitochondrial impairment in CNS injury and neurodegenerative disease.

Functionalized Phenylbenzamides Inhibit Aquaporin-4 Reducing Cerebral Edema and Improving Outcome in Two Models of CNS Injury

Cerebral edema in ischemic stroke can lead to increased intracranial pressure, reduced cerebral blood flow and neuronal death. Unfortunately, current therapies for cerebral edema are either ineffective or highly invasive. During the development of cytotoxic and subsequent ionic cerebral edema water enters the brain by moving across an intact blood brain barrier and through aquaporin-4 (AQP4) at astrocyte endfeet. Using AQP4-expressing cells, we screened small molecule libraries for inhibitors that reduce AQP4-mediated water permeability. Additional functional assays were used to validate AQP4 inhibition and identified a promising structural series for medicinal chemistry. These efforts improved potency and revealed a compound we designated AER-270, N-[3,5-bis (trifluoromethyl)phenyl]-5-chloro-2-hydroxybenzamide. AER-270 and a prodrug with enhanced solubility, AER-271 2-{[3,5-Bis(trifluoromethyl) phenyl]carbamoyl}-4-chlorophenyl dihydrogen phosphate, improved neurological outcome and reduced swelling in two models of CNS injury complicated by cerebral edema: water intoxication and ischemic stroke modeled by middle cerebral artery occlusion.

Activation of 5-HT1A Receptors Promotes Retinal Ganglion Cell Function by Inhibiting the cAMP-PKA Pathway to Modulate Presynaptic GABA Release in Chronic Glaucoma.

Serotonin (5-hydroxytryptamine, 5-HT) receptor agonists are neuroprotective in CNS injury models. However, the neuroprotective functional implications and synaptic mechanism of 8-hydroxy-2- (di-n-propylamino) tetralin (8-OH-DPAT), a serotonin receptor (5-HT1A) agonist, in an adult male Wistar rat model of chronic glaucoma model remain unknown. We found that ocular hypertension decreased 5-HT1A receptor expression in rat retinas because the number of retinal ganglion cells (RGCs) was significantly reduced in rats with induced ocular hypertension relative to that in control retinas and 8-OH-DPAT enhanced the RGC viability. The protective effects of 8-OH-DPAT were blocked by intravitreal administration of the selective 5-HT1A antagonist WAY-100635 or the selective GABAA receptor antagonist SR95531. Using patch-clamp techniques, spontaneous and miniature GABAergic IPSCs (sIPSCs and mIPSCs, respectively) of RGCs in rat retinal slices were recorded. 8-OH-DPAT significantly increased the frequency and amplitude of GABAergic sIPSCs and mIPSCs in ON- and OFF-type RGCs. Among the signaling cascades mediated by the 5-HT1A receptor, the role of cAMP-protein kinase A (PKA) signaling was investigated. The 8-OH-DPAT-induced changes at the synaptic level were enhanced by PKA inhibition by H-89 and blocked by PKA activation with bucladesine. Furthermore, the density of phosphorylated PKA (p-PKA)/PKA was significantly increased in glaucomatous retinas and 8-OH-DPAT significantly decreased p-PKA/PKA expression, which led to the inhibition of PKA phosphorylation upon relieving neurotransmitter GABA release. These results showed that the activation of 5-HT1A receptors in retinas facilitated presynaptic GABA release functions by suppressing cAMP-PKA signaling and decreasing PKA phosphorylation, which could lead to the de-excitation of RGC circuits and suppress excitotoxic processes in glaucoma.SIGNIFICANCE STATEMENT We found that serotonin (5-HT) receptors in the retina (5-HT1A receptors) were downregulated after intraocular pressure elevation. Patch-clamp recordings demonstrated differences in the frequencies of miniature GABAergic IPSCs (mIPSCs) in ON- and OFF-type retinal ganglion cells (RGCs) and RGCs in normal and glaucomatous retinal slices. Therefore, phosphorylated protein kinase A (PKA) inhibition upon release of the neurotransmitter GABA was eliminated by 8-hydroxy-2- (di-n-propylamino) tetralin (8-OH-DPAT), which led to increased levels of GABAergic mIPSCs in ON- and OFF-type RGCs, thus enhancing RGC viability and function. These protective effects were blocked by the GABAA receptor antagonist SR95531 or the 5-HT1A antagonist WAY-100635. This study identified a novel mechanism by which activation of 5-HT1A receptors protects damaged RGCs via the cAMP-PKA signaling pathway that modulates GABAergic presynaptic activity.

Establishment of Aromatic Pairs at the Surface of Chondroitinase ABC I: the Effect on Activity and Stability.

Removal of chondroitin sulfate glycosaminoglycan (GAG) chains with chondroitinase ABC I (chABC I) in CNS injury models promotes both saxon regeneration and plasticity. It has been suggested that direct interaction between an aromatic pair appears to contribute about - 1.3kcal/mol to the stability of a folded protein, so introducing an aromatic pair by point mutation might increase the enzyme activity and thermal stability as in the case of mesophilic xylanase, although using this approach destabilized T4 lysozyme. In this study, we used site-directed mutagenesis to investigate the effect of new aromatic pairs on activity and stability of chABC I. We replaced Ile295, Ser581, and Gly730 adjacent to pre-existing aromatic residues with Tyr to obtain new aromatic pairs, i.e., Tyr295/His372, Tyr576/Tyr581, and Tyr623/Tyr730. Results showed that Km values of S581Y and G730Y variants decreased relative to wild-type enzyme while their catalytic efficiency (kcat/Km) increased but I295Y variant was inactive. Also, long-term and thermal stability of the active mutants was decreased. Fluorescence and circular dichroism studies showed that these mutations resulted in a more flexible enzyme structures: a finding which was confirmed by thermal and limited proteolytic studies. In conclusion, the activity of chABC I can be improved by introducing appropriate aromatic pairs at the enzyme surface. This approach did not provide any promising results regarding the enzyme stability.

2-AG limits Theiler's virus induced acute neuroinflammation by modulating microglia and promoting MDSCs.

The innate immune response is mediated by primary immune modulators such as cytokines and chemokines that together with immune cells and resident glia orchestrate CNS immunity and inflammation. Growing evidence supports that the endocannabinoid 2-arachidonoylglycerol (2-AG) exerts protective actions in CNS injury models. Here, we used the acute phase of Theiler's virus induced demyelination disease (TMEV-IDD) as a model of acute neuroinflammation to investigate whether 2-AG modifies the brain innate immune responses to TMEV and CNS leukocyte trafficking. 2-AG or the inhibition of its hydrolysis diminished the reactivity and number of microglia at the TMEV injection site reducing their morphological complexity and modulating them towards an anti-inflammatory state via CB2 receptors. Indeed, 2-AG dampened the infiltration of immune cells into the CNS and inhibited their egress from the spleen, resulting in long-term beneficial effects at the chronic phase of the disease. Intriguingly, it is not a generalized action over leukocytes since 2-AG increased the presence and suppressive potency of myeloid derived suppressor cells (MDSCs) in the brain resulting in higher apoptotic CD4+ T cells at the injection site. Together, these data suggest a robust modulatory effect in the peripheral and central immunity by 2-AG and highlight the interest of modulating endogenous cannabinoids to regulate CNS inflammatory conditions.

Neural Stem Cells Derived from Human-Induced Pluripotent Stem Cells and Their Use in Models of CNS Injury.

Induced pluripotent stem (iPS) cells are derived from differentiated cells by different reprogramming techniques, by introducing specific transcription factors responsible for pluripotency. Induced pluripotent stem cells can serve as an excellent source for differentiated neural stem/progenitor cells (NSCs/NPs). Several methods and protocols are utilized to create a robust number of NSCs/NPs without jeopardizing the safety issues required for in vivo applications. A variety of disease-specific iPS cells have been used to study nervous system diseases. In this chapter, we will focus on some of the derivation and differentiation approaches and the application of iPS-NPs in the treatment of spinal cord injury and stroke.

A TSPO ligand attenuates brain injury after intracerebral hemorrhage.

Intracerebral hemorrhage (ICH) is a devastating disease without effective treatment. After ICH, the immediate infiltration of leukocytes and activation of microglia are accompanied by a rapid up-regulation of the 18-kDa translocator protein (TSPO). TSPO ligands have shown anti-inflammatory and neuroprotective properties in models of CNS injury. In this study, we determined the impact of a TSPO ligand, etifoxine, on brain injury and inflammation in 2 mouse models of ICH. TSPO was up-regulated in Iba1+ cells from brains of patients with ICH and in CD11b+CD45int cells from mice subjected to collagenase-induced ICH. Etifoxine significantly reduced neurodeficits and perihematomal brain edema after ICH induction by injection of either autologous blood or collagenase. In collagenase-induced ICH mice, the protection of etifoxine was associated with reduced leukocyte infiltration into the brain and microglial production of IL-6 and TNF-α. Etifoxine improved blood–brain barrier integrity and diminished cell death. Notably, the protective effect of etifoxine was abolished in mice depleted of microglia by using a colony-stimulating factor 1 receptor inhibitor. These results indicate that the TSPO ligand etifoxine attenuates brain injury and inflammation after ICH. TSPO may be a viable therapeutic target that requires further investigations in ICH.—Li, M., Ren, H., Sheth, K. N., Shi, F.-D., Liu, Q. A TSPO ligand attenuates brain injury after intracerebral hemorrhage.

Prostaglandin E2 Produced by Alginate-Encapsulated Mesenchymal Stromal Cells Modulates the Astrocyte Inflammatory Response.

Astroglia are well known for their role in propagating secondary injury following brain trauma. Modulation of this injury cascade, including inflammation, is essential to repair and recovery. Mesenchymal stromal cells (MSCs) have been demonstrated as trophic mediators in several models of secondary CNS injury, however, there has been varied success with the use of direct implantation due to a failure to persist at the injury site. To achieve sustained therapeutic benefit, we have encapsulated MSCs in alginate microspheres and evaluated the ability of these encapsulated MSCs to attenuate neuro-inflammation. In this study, astroglial cultures were administered lipopolysaccharide (LPS) to induce inflammation and immediately co-cultured with encapsulated or monolayer human MSCs. Cultures were assayed for the pro-inflammatory cytokine tumor necrosis factor alpha (TNF-α) produced by astroglia, MSC-produced prostaglandin E2, and expression of neurotrophin-associated genes. We found that encapsulated MSCs significantly reduced TNF-α produced by LPS-stimulated astrocytes, more effectively than monolayer MSCs, and this enhanced benefit commences earlier than that of monolayer MSCs. Furthermore, in support of previous findings, encapsulated MSCs constitutively produced high levels of PGE2, while monolayer MSCs required the presence of inflammatory stimuli to induce PGE2 production. The early, constitutive presence of PGE2 significantly reduced astrocyte-produced TNF-α, while delayed administration had no effect. Finally, MSC-produced PGE2 was not only capable of modulating inflammation, but appears to have an additional role in stimulating astrocyte neurotrophin production. Overall, these results support the enhanced benefit of encapsulated MSC treatment, both in modulating the inflammatory response and providing neuroprotection.

Antagonization of the Nogo-Receptor 1 Enhances Dopaminergic Fiber Outgrowth of Transplants in a Rat Model of Parkinson's Disease.

Intrastriatal transplantation of fetal human ventral mesencephalic dopaminergic neurons is an experimental therapy for patients suffering from Parkinson’s disease. The success of this approach depends on several host brain parameters including neurotrophic factors and growth inhibitors that guide survival and integration of transplanted neurons. While the potential of neurotrophic factors has been extensively investigated, repression of growth inhibitors has been neglected, despite the significant effects reported in various CNS injury models. Recently, we demonstrated that infusion of neutralizing antibodies against Nogo-A into the lateral ventricles of hemi-parkinsonian rats significantly enhanced graft function. Since the Nogo-receptor 1 also interacts with other neurite growth inhibitors, we investigated whether a direct antagonization of the receptor would result in more robust effects. Therefore, rats with unilateral striatal 6-hydroxydopamine lesions were grafted with ventral mesencephalic tissue in combination with intraventricular infusions of the Nogo-receptor 1 antagonist NEP1-40. Transplanted rats receiving saline infusions served as controls. To test whether NEP1-40 treatment alone affects the remaining dopaminergic striatal fibers, rats with unilateral striatal 6-hydroxydopamine lesions were infused with NEP1-40 or saline without receiving a transplant. Motor behavior was assessed prior to the lesion as well as prior and 1, 3, and 5 weeks after the transplantations. At the end of the experimental period the number of graft-derived dopaminergic fibers growing into the host brain, the number of surviving dopaminergic neurons and graft volume were analyzed. In rats without a transplant, the density of dopaminergic fibers in the striatum was analyzed. We detected that NEP1-40 treatment significantly enhanced graft-derived dopaminergic fiber outgrowth as compared to controls while no effects were detected for graft volume and survival of grafted dopaminergic neurons. Notably, the enhanced dopaminergic fiber outgrowth was not sufficient to improve the functional recovery as compared to controls. Moreover, NEP1-40 infusions in hemi-parkinsonian rats without a transplant did not result in enhanced striatal dopaminergic fiber densities and consequently did not improve behavior. In sum, our findings demonstrate that antagonization of the Nogo-receptor 1 has the capacity to support the engraftment of transplanted mesencephalic tissue in an animal model of Parkinson’s disease.

An immunoaffinity-based method for isolating ultrapure adult astrocytes based on ATP1B2 targeting by the ACSA-2 antibody

Astrocytes are a major cell type in the mammalian CNS. Astrocytes are now known to play a number of essential roles in processes including synapse formation and function, as well as blood-brain barrier formation and control of cerebral blood flow. However, our understanding of the molecular mechanisms underlying astrocyte development and function is still rudimentary. This lack of knowledge is at least partly due to the lack of tools currently available for astrocyte biology. ACSA-2 is a commercially available antibody originally developed for the isolation of astrocytes from young postnatal mouse brain, using magnetic cell-sorting methods, but its utility in isolating cells from adult tissue has not yet been published. Using a modified protocol, we now show that this tool can also be used to isolate ultrapure astrocytes from the adult brain. Furthermore, using a variety of techniques (including single-cell sequencing, overexpression and knockdown assays, immunoblotting, and immunohistochemistry), we identify the ACSA-2 epitope for the first time as ATP1B2 and characterize its distribution in the CNS. Finally, we show that ATP1B2 is stably expressed in multiple models of CNS injury and disease. Hence, we show that the ACSA-2 antibody possesses the potential to be an extremely valuable tool for astrocyte research, allowing the purification and characterization of astrocytes (potentially including injury and disease models) without the need for any specialized and expensive equipment. In fact, our results suggest that ACSA-2 should be a first-choice method for astrocyte isolation and characterization.

Effect of granulocyte colony-stimulating factor on the cochlear nuclei after creation of a partial nerve lesion: an experimental study in rats.

OBJECTIVE The risk of injury of the cochlear nerve during angle (CPA) surgery is high. Granulocyte colony-stimulating factor (G-CSF) has been found in various experimental models of peripheral and CNS injury to have a neuroprotective effect by inhibiting apoptosis and inflammation. However, to the authors' knowledge, the influence of G-CSF on cochlear nerve regeneration has not been reported. This study investigated the neuroprotective effect of G-CSF after a partial cochlear nerve lesion in rats. METHODS A lesion of the right cochlear nerve in adult male Sprague-Dawley rats was created using a water-jet dissector with a pressure of 8 bar. In the first group (G-CSF-post), G-CSF was administrated on Days 1, 3, and 5 after the surgery. The second group (G-CSF-pre/post) was treated with G-CSF 1 day before and 1, 3, and 5 days after applying the nerve injury. The control group received sodium chloride after nerve injury at the various time points. Brainstem auditory evoked potentials (BAEPs) were measured directly before and after nerve injury and on Days 1 and 7 to evaluate the acoustic function of the cochlear nerve. The animals were sacrificed 1 week after the operation, and their brains were fixed in formalin. Nissl staining of the cochlear nuclei was performed, and histological sections were analyzed with a light microscope and an image-processing program. The numbers of neurons in the cochlear nuclei were assessed. RESULTS The values for Waves 2 and 4 of the BAEPs decreased abruptly in all 3 groups in the direct postoperative measurement. Although the amplitude in the control group did not recover, it increased in both treatment groups. According to 2-way ANOVA, groups treated with G-CSF had a significant increase in BAEP Wave II amplitudes on the right side (p = 0.0401) after the applied cochlear nerve injury. With respect to Wave IV, a trend toward better recovery in the G-CSF groups was found, but this difference did not reach statistical significance. In the histological analysis, higher numbers of neurons were found in the G-CSF groups. In the statistical analysis, the difference in the numbers of neurons between the control and G-CSF-post groups reached significance (p = 0.0086). The difference in the numbers of neurons between the control and G-CSF-pre/post groups and between the G-CSF-post and G-CSF-pre/post groups did not reach statistical significance. CONCLUSIONS The use of G-CSF improved the function of the eighth cranial nerve and protected cochlear nucleus cells from destruction after a controlled partial injury of the nerve. These findings might be relevant for surgery that involves CPA tumors. The use of G-CSF in patients with a lesion in the CPA might improve postoperative outcomes.

Abstract TP84: Comparative Analysis of Genetic and Pharmacological Inhibition of Sirt3 in Post Ischemic Injury

Background: Sirtuin3 (Sirt3), NAD+-dependent deacetylase, regulates several key proteins in both mitochondria and nucleus. The majority of early studies have shown neuroprotective effects of Sirt3 activation in various models of CNS injury. Recently, it was found that genetic loss of Sirt3 is beneficial in ischemia/reperfusion (I/R) injury. Therefore, this study was designed to explore the effects of both genetic and pharmacological inhibition of Sirt3 in a middle cerebral artery occlusion (MCAo) model of stroke. Methods: Focal cerebral ischemia was induced by transient right MCAo for 60min followed by 0, 6, 24 and 72 hrs of reperfusion. A 72hrs post ischemic injury time point was chosen to measure infarction in various treatment groups. The Sirt3 inhibitor AGK7 (sc- 204281 Santa cruz,) that selectively inhibits Sirt3 over Sirt1 and Sirt2, was given as single intraperitoneal injection (0.15, 0.5 and 1.5mg/kg) 3hrs after stroke onset. Activity or expression of Sirt3, was examined with Sirt3 activity kit or western blot using anti-Sirt3, anti-acetylated lysine antibodies. Results: Sirt3 knockout mice showed a significant reduction in hemispheric infarct volume compared to WT littermate controls (37.72±5.21 vs 52.8±6.76, p<0.05). In the pharmacological study, a dose of 0.5 mg/kg i.p of inhibitor showed significant reduction in Sirt3 activity and but had no neuroprotective effect. Furthermore, a much higher dose (1.5mg/kg i.p) did not show any additional inhibitory effect on sirt3 activity but unexpectedly increased infarct volume (51.8±6.87 vs. 62.56± 7.89, P<0.05) and hemorrhagic transformation. Expression studies showed no overall change in Sirt3 expression in whole cell lysates at 0, 6, 24 or 72 hrs after reperfusion; however the expression pattern and activity varied with time in different subcellular compartments. Summary and Conclusions: Changes in subcellular translocation and activity of Sirt3 after I/R stress suggests it important role in target protein deacetylation in stroke pathophysiology. Surprisingly, genetic deletion but not pharmacological inhibition led to neuroprotection, indicating the need to carefully examine target protein by both genetic and pharmacological approaches.