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Aims Naltrexone is a mu opioid receptor (MOR) antagonist used to treat drug dependence in patients. Previous reports indicated that MOR antagonists reduced neurodegeneration and inflammation after brain injury. The purpose of this study was to evaluate the neuroprotective effect of naltrexone in cell culture and a mouse model of traumatic brain injury (TBI). Methods The neuroprotective effect of naltrexone was examined in primary cortical neurons co‐cultured with BV2 microglia. Controlled cortical impact (CCI) was delivered to the left cerebral cortex of adult male MOR wild‐type (WT) and knockout (KO) mice. Naltrexone was given daily for 4 days, starting from day 2 after lesioning. Locomotor activity was evaluated on day 5 after the CCI. Brain tissues were collected for immunostaining, Western, and qPCR analysis. Results Glutamate reduced MAP2 immunoreactivity (‐ir), while increased IBA1‐ir in neuron/BV2 co‐culture; both responses were antagonized by naltrexone. TBI significantly reduced locomotor activity and increased the expression of IBA1, iNOS, and CD4 in the lesioned cortex. Naltrexone significantly and equally antagonized the motor deficits and expression of IBA1 and iNOS in WT and KO mice. TBI‐mediated CD4 protein production was attenuated by naltrexone in WT mice, but not in KO mice. Conclusion Naltrexone reduced TBI‐mediated neurodegeneration and inflammation in MOR WT and KO mice. The protective effect of naltrexone involves non‐MOR and MOR mechanisms., Naltrexone reduced glutamate or TBI‐mediated neurodegeneration and inflammation in MOR KO and WT mice.

2'-Fucosyllactose (2'-FL) is a major oligosaccharide in human milk and is present at trace levels in cow milk. 2'-FL reduces inflammation in the gastrointestinal tract. Its action in the central nervous system has not been well characterized. The purpose of this study is to determine 2'-FL-mediated neural protection and repair in culture and stroke brain. In rat primary cortical neuronal cultures, 2'-FL significantly antagonized N-methyl-D-aspartate (NMDA) or glutamate-mediated changes in ATP production, MAP2 immunoreactivity, and TUNEL. The influx of Ca++ (Ca++i) was examined in primary cortical neurons expressing GCaMP5, an endogenous calcium probe. NMDA increased Ca++i; 2'-FL significantly attenuated this reaction. In a rat middle cerebral artery occlusion model of stroke, we found that intracerebroventricular pretreatment or oral posttreatment with 2'-FL significantly reduced brain infarction, mitigated microglial activation, improved locomotor activity, and upregulated brain-derived neurotrophic factor (BDNF) expression. Post-stroke delivery of 2'-FL increased bromodeoxyuridine (BrdU) labeling in the perilesioned area. These BrdU cells co-expressed NeuN, or nestin, or GFAP. Using subventricular Matrigel cultures, we demonstrated that 2'-FL increased cell migration from subventricular zone explant. This response was reduced by anti-BDNF blocking antibody. In conclusion, our data suggest that 2'-FL has neuroprotective action through inhibition of Ca++i, inflammation, and apoptosis. Posttreatment with 2'-FL facilitates neural repair in stroke brain.

Intracerebral hemorrhage (ICH) occurs when brain blood vessels rupture, causing inflammation and cell death. 2-Fucosyllactose (2FL), a human milk oligosaccharide, has potent antiapoptotic and anti-inflammatory effects. The purpose of this study was to examine the protective effect of 2FL in cellular and rodent models of ICH. Hemin was added to a primary rat cortical neuronal and BV2 microglia coculture to simulate ICH in vitro. IBA1 and MAP2 immunoreactivities were used to determine inflammation and neuronal survival. Hemin significantly increased IBA1, while it reduced MAP2 immunoreactivity. 2FL significantly antagonized both responses. The protective effect of 2FL was next examined in a rat ICH model. Intracerebral administration of type VII collagenase reduced open-field locomotor activity. Early post-treatment with 2FL significantly improved locomotor activity. Brain tissues were collected for immunohistochemistry and qRT-PCR analysis. 2FL reduced IBA1 and CD4 immunoreactivity in the lesioned striatum. 2FL downregulated the expression of ER stress markers (PERK and CHOP), while it upregulated M2 macrophage markers (CD206 and TGFβ) in the lesioned brain. Taken together, our data support that 2FL has a neuroprotective effect against ICH through the inhibition of neuroinflammation and ER stress. 2FL may have clinical implications for the treatment of ICH.

A major limitation with cell transplantation in patients is the unimpressive number of cells survived. The death of grafted cells involves apoptosis and immunorejection. In this review, we encapsulate the recent preclinical development that improves the survival of grafted cells and mitigates the immunorejection of human-induced pluripotent stem cells (iPSCs) through co-grating nanoparticles-containing cyclosporine A (NanoCsA) in hemiparkinsonian rats. The study supported the notion that NanoCsA allows for long-lasting CsA discharge and limits immunorejection of human iPSC xenograft in a 6-hydroxydopamine Parkinson's disease rat model.

Increasing evidence has supported that transplantation of human stem cells induces neuroprotective and reparative effects in animal models of Parkinson’s disease (PD). However, without systemic immunosuppressive therapy, most of these grafted cells are rejected by the hosts. Long term and systemic injection of cyclosporine-A (CsA) is required to maintain the survival of grafted cells. The purpose this study is to examine a new treatment strategy to suppress the immunorejection by locally co-grafting of polylactic/glycolic acid nanoparticles containing CsA (NanoCsA) with differentiated human induced pluripotent stem cells (iPSCs). In the in vitro media, NanoCsA provided sustained release of CsA for >6 weeks. The differentiated human iPSCs were co-grafted with NanoCsA or NanoVeh (nanoparticle without CsA) to the striatum of unilaterally 6-hydroxydopamine -lesioned rats. NanoCsA/iPSCs co-graft significantly improved locomotor activity compared to NanoVeh/iPSCs co-grafts or iPSC grafts + sytemic CsA at 1 month after transplantation. Brain tissues were collected for measurements of tyrosine hydroxylase (TH) and human marker Stem121 immunoreactivity. Cografting with NanoCsA/iPSCs, compared to NanoVeh/iPSCs, significantly increased TH and Stem121 immunoreactivity as well as tumor formation in the lesioned striatum. Taken together, our study supports that NanoCsA provides long-lasting CsA release and reduces immunorejection of human iPSCs xenograft in a 6-hydroxydopamine rat model of PD.

Methamphetamine (Meth) is a widely abused stimulant. High-dose Meth induces degeneration of dopaminergic neurons through p53-mediated apoptosis. A recent study indicated that treatment with the p53 inhibitor, pifithrin-alpha (PFT-α), antagonized Meth-mediated behavioral deficits in mice. The mechanisms underpinning the protective action of PFT-α against Meth have not been identified, and hence, their investigation is the focus of this study. Primary dopaminergic neuronal cultures were prepared from rat embryonic ventral mesencephalic tissue. High-dose Meth challenge reduced tyrosine hydroxylase immunoreactivity and increased terminal deoxynucleotidyl transferase-mediated dNTP nick-end labeling (TUNEL) labeling. PFT-α significantly antagonized these responses. PFT-α also reduced Meth-activated translocation of p53 to the nucleus, an initial step before transcription. Previous studies have indicated that p53 can also activate cell death through transcription-independent pathways. We found that PFT-α attenuated endoplasmic reticulum (ER) stressor thapsigargin (Tg)-mediated loss of dopaminergic neurons. ER stress was further monitored through the release of Gaussia luciferase (GLuc) from SH-SY5Y cells overexpressing GLuc-based Secreted ER Calcium-Modulated Protein (GLuc-SERCaMP). Meth or Tg significantly increased GLuc release in to the media, with PFT-α significantly reducing GLuc release. Additionally, PFT-α significantly attenuated Meth-induced CHOP expression. In conclusion, our data indicate that PFT-α is neuroprotective against Meth-mediated neurodegeneration via transcription-dependent nuclear and -independent cytosolic ER stress pathways.

Transplantation of human bone marrow mesenchymal stem cells (hMSCs) stands as a potent stroke therapy, but its exact mechanism remains unknown. This study investigated the anti-apoptotic mechanisms by which hMSCs exert neuroprotective effects on cerebral ischemia. Primary mixed cultures of rat neurons and astrocytes were cultured and exposed to oxygen-glucose deprivation. A two-hour period of “reperfusion” in standard medium and normoxic conditions was allowed and immediately followed by hMSCs and/or Bcl-2 antibody treatment. Cell viability of primary rat neurons and astrocytes was determined by 3-(4,5-dimethylthianol-2-yl)-2,5 diphenyl tetrazolium bromide and trypan blue exclusion methods. hMSC survival and differentiation were characterized by immunocytochemistry, while the concentration of Bcl-2 in the supernatant was measured by enzyme-linked immunosorbent assay to reveal the secretory anti-apoptotic function of hMSCs. Cultured hMSCs expressed embryonic-like stem cell phenotypic markers CXCR4, Oct4, SSEA4, and Nanog, as well as immature neural phenotypic marker Nestin. Primary rat neurons and astrocytes were protected from oxygen-glucose deprivation by hMSCs, which was antagonized by the Bcl-2 antibody. However, Bcl-2 levels in the supernatants did not differ between hMSC- and non-treated cells exposed to oxygen-glucose deprivation. Neuroprotective effects of hMSCs against cerebral ischemia were partially mediated by the anti-apoptotic mechanisms. However, further studies are warranted to fully elucidate this pathway.

Background We previously demonstrated that subcutaneous administration of PT320, a sustained-release (SR) form of exendin-4, resulted in the long-term maintenance of steady-state exenatide (exendin-4) plasma and target levels in 6-hydroxydopamine (6-OHDA)-pretreated animals. Additionally, pre- or post-treatment with PT320 mitigated the early stage of 6-OHDA-induced dopaminergic neurodegeneration. The purpose of this study was to evaluate the effect of PT320 on L-3,4-dihydroxyphenylalanine (L-DOPA)-induced abnormal involuntary movements (AIMs) in the rat 6-OHDA model of Parkinson's disease. Methods Adult male Sprague-Dawley rats were unilaterally lesioned in the right medial forebrain bundle by 6-OHDA. L-DOPA and benserazide were given daily for 22 days, starting from 4 weeks after lesioning. PT320 was co-administered weekly for 3 weeks. AIM was evaluated on days 1, 16, and 22 after initiating L-DOPA/benserazide + PT320 treatment. Brain tissues were subsequently collected for HPLC measurements of dopamine (DA) and metabolite concentrations. Results L-DOPA/benserazide increased AIMs of limbs and axial as well as the sum of all dyskinesia scores (ALO) over 3 weeks. PT320 significantly reduced the AIM scores of limbs, orolingual, and ALO. Although PT320 did not alter DA levels in the lesioned striatum, PT320 significantly attenuated 6-OHDA-enhanced DA turnover. Conclusion PT320 attenuates L-DOPA/benserazide-induced dyskinesia in a 6-OHDA rat model of PD and warrants clinical evaluation to mitigate Parkinson's disease in humans.

Background Accumulation of α-synuclein (αSyn) in the dopaminergic neurons is a common pathology seen in patients with Parkinson's disease (PD). Overproduction of αSyn potentiates the formation of oligomeric αSyn aggregates and enhances dopaminergic neuron degeneration. Downregulating intracellular monomeric αSyn prevents the formation of αSyn oligomers and is a potential therapeutic strategy to attenuate the progression of PD. Objective The purpose of this study is to investigate the efficacy of gene delivery of αSyn-specific single-chain antibodies in vitro and in vivo. Methods and results The plasmids for αSyn and selective antibodies (NAC32, D10, and VH14) were constructed and were transfected to HEK293 and SH-SY5Y cells. Co-expression of αSyn with NAC32, but not D10 or VH14, profoundly downregulated αSyn protein, but not αSyn mRNA levels in these cells. The interaction of αSyn and NAC32 antibody was next examined in vivo. Adeno-associated virus (AAV)-αSyn combined with AAV-NAC32 or AAV-sc6H4 (a negative control virus) were stereotactically injected into the substantia nigra of adult rats. AAV-NAC32 significantly reduced AAV-encoded αSyn levels in the substantia nigra and striatum and increased tyrosine hydroxylase immunoreactivity in the striatum. Also, in the animals injected with AAV-NAC32 alone, endogenous αSyn protein levels were significantly downregulated in the substantia nigra. Conclusion Our data suggest that AAV-mediated gene transfer of NAC32 is a feasible approach for reducing the expression of target αSyn protein in brain.

Stroke induces not only cell death but also neurorepair. De novo neurogenesis has been found in the subventricular zone of the adult mammalian brain days after stroke. Most of these newly generated cells die shortly after the insult. Recent studies have shown that pharmacological manipulation can improve the survival of endogenous neuroprogenitor cells and neural regeneration in stroke rats. As these drugs target the endogenous reparative processes that occur days after stroke, they may provide a prolonged window for stroke therapy. Here, we discuss endogenous neurogenesis-enhancing drugs and review the general status of stroke therapeutics in evaluating the field of pharmacotherapy for stroke.

Several single incretin receptor agonists that are approved for the treatment of type 2 diabetes mellitus (T2DM) have been shown to be neuroprotective in cell and animal models of neurodegeneration. Recently, a synthetic dual incretin receptor agonist, nicknamed "twincretin," was shown to improve upon the metabolic benefits of single receptor agonists in mouse and monkey models of T2DM. In the current study, the neuroprotective effects of twincretin are probed in cell and mouse models of mild traumatic brain injury (mTBI), a prevalent cause of neurodegeneration in toddlers, teenagers and the elderly. Twincretin is herein shown to have activity at two different receptors, dose-dependently increase levels of intermediates in the neurotrophic CREB pathway and enhance viability of human neuroblastoma cells exposed to toxic concentrations of glutamate and hydrogen peroxide, insults mimicking the inflammatory conditions in the brain post-mTBI. Additionally, twincretin is shown to improve upon the neurotrophic effects of single incretin receptor agonists in these same cells. Finally, a clinically translatable dose of twincretin, when administered post-mTBI, is shown to fully restore the visual and spatial memory deficits induced by mTBI, as evaluated in a mouse model of weight drop close head injury. These results establish twincretin as a novel neuroprotective agent and suggest that it may improve upon the effects of the single incretin receptor agonists via dual agonism.

Cell death and neurogenesis have been examined after stroke in the subventricular zone of the adult mammalian brain. New research focuses on the use of drugs to improve the viability of neural progenitor cells in rats after stroke. The aim of the drugs is to lengthen the timeframe for stroke therapy by targeting the endogenous repair mechanism that follows injury. In this paper, we look at the broad state of stroke therapy to assess the effectiveness of endogenous neurogenesis-enhancing drugs on stroke. This paper is a review article. Referred literature in this paper has been listed in the reference section. The data sets supporting the conclusions of this article are available online by searching various databases, including PubMed. Some original points in this article come from the laboratory practice in our research center and the authors' experiences.

Accumulation of α-Synuclein (αSyn) in nigral dopaminergic neurons is commonly seen in patients with Parkinson′s disease (PD). We recently reported that transduction of intracellular single-chain intrabody targeting the 53–87 amino acid residues of human αSyn by recombinant adeno associated viral vector (AAV-NAC32) downregulated αSyn protein in SH-SY5Y cells and rat brain. This study characterizes the behavioral phenotype and dopaminergic protection in animals receiving AAV-NAC32. Our results show that adult DAT-Cre rats selectively overexpress αSyn in nigra dopaminergic neurons after local administration of AAV-DIO-αSyn. These animals develop PD-like phenotype, including bradykinesia and loss of tyrosine hydroxylase (TH) immunoreactivity in substantia nigra pars compacta dorsal tier (SNcd). An injection of AAV-NAC32 to nigra produces a selective antibody against αSyn and normalizes the behavior. AAV-NAC32 significantly increases TH, while reduces αSyn immunoreactivity in SNcd. Altogether, our data suggest that an AAV-mediated gene transfer of NAC32 antibody effectively antagonizes αSyn-mediated dopaminergic degeneration in nigra, which may be a promising therapeutic candidate for synucleinopathy or PD.

Amphetamine-type stimulants have become important and popular abused drugs worldwide. Methamphetamine (Meth) sensitization, characterized by a progressive increase in behavioral responses after repeated administration, has been reported in rodents and patients. This behavioral effect has been used as a laboratory model to study drug addiction and schizophrenia. The mesolimbic dopaminergic pathway plays a significant role in the development of Meth behavioral sensitization. Previous studies have reported that the ablation of nucleus accumbens (NAc) by electrolytic or thermal lesioning attenuates addictive behavior to opioids in animals. However, these studies were only conducted in opioid addictive rodents. Furthermore, these ablation procedures also damaged the non-dopaminergic neurons and fibers passing through the NAc. The purpose of this study was to examine the therapeutic effect of NAc lesioning by a selective dopaminergic toxin in Meth-sensitized animals. Adult mice received repeated administration of Meth for 7 days. Open-field locomotor activity and stereotype behavior were significantly increased after Meth treatment, suggesting behavior sensitization. A partial lesion of dopaminergic terminals was made through stereotaxic administration of dopaminergic toxin 6-hydroxydopamine (6-OHDA) to the NAc in the Meth -sensitized mice. Meth behavioral sensitization was significantly antagonized after the lesioning. Brain tissue was collected for qRT-PCR analysis. Repeated administration of Meth increased the expression of tyrosine hydroxylase (TH), BDNF, and Shati, a marker for Meth sensitization, in the NAc. Treatment with 6-OHDA significantly antagonized the upregulation of TH and Shati. Taken together, these data suggest that local administration of 6-OHDA mitigated Meth sensitization in chronic Meth-treated animals. Our data support a new surgical treatment strategy for Meth abuse.

Intracerebral hemorrhage (ICH) is a major cause of stroke, with high mortality and morbidity. There is no effective pharmacological therapy for ICH. Previous studies have indicated that CXCR4 antagonists reduced microglia activation, attenuated infiltration of T cells, and improved functional recovery in ischemic stroke animals. The interaction of CXCR4 antagonists and ICH has not been characterized. The purpose of this study is to examine the neuroprotective action of a novel CXCR4 antagonist CX807 against ICH. In primary cortical neuronal and BV2 microglia co-culture, CX807 reduced glutamate-mediated neuronal loss and microglia activation. Adult rats were locally administered with collagenase VII to induce ICH. CX807 was given systemically after the ICH. Early post-treatment with CX807 improved locomotor activity in ICH rats. Brain tissues were collected for qRTPCR and histological staining. ICH upregulated the expression of CXCR4, CD8, TNF&alpha, IL6, and TLR4. The immunoreactivity of IBA1 and CD8, as well as TUNEL labeling, were enhanced in the perilesioned area. CX807 significantly mitigated these responses. In conclusion, our data suggest that CX807 is neuroprotective and anti-inflammatory against ICH. CX807 may have clinical implications for the treatment of hemorrhagic stroke.

Summary Acetylcholinesterase (AChE) inhibitors have protective and anti-inflammatory actions against brain injury, mediated by nicotinic α7 cholinergic receptor activation. The use of AChE inhibitors in patients is limited by systemic cholinergic side effects. Posiphen, a stereoisomer of the AChE inhibitor Phenserine, lacks AChE inhibitor activity. The purpose of this study is to determine the protective effect of Posiphen in cellular and animal models of stroke. Both Posiphen and Phenserine reduced glutamate-mediated neuronal loss in co-cultures of primary cortical cells and microglia. Phenserine-, but not Posiphen-, mediated neuroprotection was diminished by the nicotinic α7 receptor antagonist methyllycaconitine. Posiphen antagonized NMDA-mediated Ca++ influx, thapsigargin-mediated neuronal loss and ER stress in cultured cells. Early post-treatment with Posiphen reduced ER stress signals, IBA1 immunoreactivity, TUNEL and infarction in the ischemic cortex, as well as neurological deficits in stroke rats. These findings indicate that Posiphen is neuroprotective against stroke through regulating Ca++i and ER stress., Graphical Abstract, Highlights • Posiphen induces protection in cell culture through noncholinergic mechanism • Posiphen attenuates glutamate-mediated Ca++i and ER stress in neuronal culture • Posiphen mitigates ER stress in stroke brain • Posiphen reduces neurodegeneration in stroke rats, Drugs; Molecular Biology; Clinical Neuroscience; Cell Biology

Wharton’s jelly-derived mesenchymal stromal cells (WJ-MSCs) have distinct immunomodulatory and protective effects against kidney, liver, or heart injury. Limited studies have shown that WJ-MSCs attenuates oxygen–glucose deprivation-mediated inflammation in hippocampal slices. The neuroprotective effect of intracerebral WJ-MSC transplantation against stroke has not been well characterized. The purpose of this study was to examine the neuroprotective effect of human WJ-MSC (hWJ-MSC) transplants in an animal model of stroke. Adult male Sprague–Dawley rats were anesthetized and placed in a stereotaxic frame. hWJ-MSCs, pre-labeled with chloromethyl benzamide 1,1’-dioctadecyl-3,3,3’3’- tetramethylindocarbocyanine perchlorate (CM-Dil), were transplanted to the right cerebral cortex at 10 min before a transient (60 min) right middle cerebral artery occlusion (MCAo). Transplantation of hWJ-MSCs significantly reduced neurological deficits at 3 and 5 days after MCAo. hWJ-MSC transplants also significantly reduced brain infarction and microglia activation in the penumbra. Grafted cells carrying CM-Dil fluorescence were identified at the grafted site in the ischemic core; these cells were mostly incorporated into ionized calcium-binding adaptor molecule (+) cells, suggesting these xenograft cells were immuno-rejected by the host. In another set of animals, hWJ-MSCs were transplanted in cyclosporine (CsA)-treated rats. hWJ-MSC transplants significantly reduced brain infarction, improved neurological function, and reduced neuroinflammation. Less phagocytosis of CM-dil-labeled grafted cells was found in the host brain after CsA treatment. Transplantation of hWJ-MSC significantly increased glia cell line-derived neurotrophic factor expression in the host brain. Taken together, our data support that intracerebral transplantation of hWJ-MSCs reduced neurodegeneration and inflammation in the stroke brain. The protective effect did not depend on the survival of grafted cells but may be indirectly mediated through the production of protective trophic factors from the transplants.

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We previously demonstrated that pretreatment with Exendin-4, a glucagon-like peptide-1 (GLP-1) receptor agonist, reduces 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) –mediated dopaminergic neurodegeneration. The use of GLP-1 or Exendin-4 for Parkinson’s disease (PD) patients is limited by their short half-lives. The purpose of this study was to evaluate a new extended release Exendin-4 formulation, PT302, in a rat model of PD. Subcutaneous administration of PT302 resulted in sustained elevations of Exendin-4 in plasma for >20 days in adult rats. To define an efficacious dose within this range, rats were administered PT302 once every 2 weeks either before or following the unilaterally 6-hydroxydopamine lesioning. Pre- and post-treatment with PT302 significantly reduced methamphetamine–induced rotation after lesioning. For animals given PT302 post lesion, blood and brain samples were collected on day 47 for measurements of plasma Exendin-4 levels and brain tyrosine hydroxylase immunoreactivity (TH-IR). PT302 significantly increased TH-IR in the lesioned substantia nigra and striatum. There was a significant correlation between plasma Exendin-4 levels and TH-IR in the substantia nigra and striatum on the lesioned side. Our data suggest that post-treatment with PT302 provides long-lasting Exendin-4 release and reduces neurodegeneration of nigrostriatal dopaminergic neurons in a 6-hydroxydopamine rat model of PD at a clinically relevant dose.

RATIONALE: Repeated administration of methamphetamine (Meth) induces behavioral sensitization which is characterized by a progressive increase in locomotor response after each injection. Previous studies have shown that Mu opioid receptors (MORs) can regulate Meth-mediated behavioral sensitization. However, the reported interactions are controversial; systemic activation of MORs either enhanced or suppressed Meth sensitization. It is possible that alteration of Meth sensitization after systemic administration of MOR ligands reflects the sum of distinct MOR reactions in multiple brain regions. OBJECTIVES: The purpose of the present study was to examine the actions of MORs on Meth sensitization after regionally selective overexpression of human MOR through an AAV6-based gene delivery system. METHOD: We demonstrated that adeno-associated virus (AAV)-MOR increased MOR immunoreactivity and binding in vitro. AAV-MOR or AAV-green fluorescent protein (GFP) was injected into the nucleus accumbens (NAc) or ventral tegmental area (VTA) of adult mice. Two weeks after viral infection, animals received Meth or saline for five consecutive days. Locomotor behavior and striatal dopamine (DA) and 3,4-dihydroxyphenylacetic acid (DOPAC) level were determined. RESULTS: Repeated administration of Meth progressively increased locomotor activity; this sensitization reaction was attenuated by intra-NAc AAV-MOR microinjections. Infusion of AAV-MOR to VTA enhanced Meth sensitization. AAV-MOR significantly enhanced DA levels in VTA after VTA infection but reduced DOPAC/DA turnover in the NAc after NAc injection. CONCLUSION: Our data suggest a differential modulation of Meth sensitization by overexpression of MOR in NAc and VTA. Regional manipulation of MOR expression through AAV may be a novel approach to control Meth abuse and psychomimetic activity.

The purpose of this study was to examine the neurorestorative effect of delayed 9 cis retinoic acid (9cRA) treatment for stroke. Adult male rats received a 90-min right distal middle cerebral artery occlusion (dMCAo). Animals were separated into two groups with similar infarction sizes, based on magnetic resonance imaging on day 2 after dMCAo. 9cRA or vehicle was given via an intranasal route daily starting from day 3. Stroke rats receiving 9cRA post-treatment showed an increase in brain 9cRA levels and greater recovery in motor function. 9cRA enhanced the proliferation of bromodeoxyuridine (+) cells in the subventricular zone (SVZ) and lesioned cortex in the stroke brain. Using subventricular neurosphere and matrigel cultures, we demonstrated that proliferation and migration of SVZ neuroprogenitor cells were enhanced by 9cRA. Our data support a delayed and non-invasive drug therapy for stroke. Intranasal 9cRA can facilitate the functional recovery and endogenous repair in the ischemic brain.

C-X-C chemokine receptor type 4 (CXCR4) is a receptor for a pleiotropic chemokine CXCL12. Previous studies have shown that the acute administration of the CXCR4 antagonist AMD3100 reduced neuroinflammation in stroke brain and mobilized bone marrow hematopoietic stem cells (HSCs). The purpose of this study was to characterize the neuroprotective and neurotrophic effect of a novel CXCR4 antagonist CX549. We demonstrated that CX549 had a higher affinity for CXCR4 and was more potent than AMD3100 to inhibit CXCL12-mediated chemotaxis in culture. CX549 effectively reduced the activation of microglia and improved neuronal survival after injury in neuron/microglia cocultures. Early poststroke treatment with CX549 significantly improved behavioral function, reduced brain infarction, and suppressed the expression of inflammatory markers. Compared to AMD3100, CX549 has a higher affinity for CXCR4, is more efficient to mobilize HSCs for transplantation, and induces behavioral improvement. Our data support that CX549 is a potent anti-inflammatory agent, is neuroprotective against ischemic brain injury, and may have clinical implications for the treatment of stroke.

In this study, methamphetamine (Meth)- and glutamate (Glu)-mediated intracellular Ca(++) (Ca(++) i) signals were examined in real time in primary cortical neurons overexpressing an intracellular Ca(++) probe, GCaMP5, by adeno-associated viral (AAV) serotype 1. Binding of Ca(++) to GCaMP increased green fluorescence intensity in cells. Both Meth and Glu induced a rapid increase in Ca(++) i, which was blocked by MK801, suggesting that Meth enhanced Ca(++) i through Glu receptor in neurons. The Meth-mediated Ca(++) signal was also blocked by Mg(++) , low Ca(++) or the L-type Ca(++) channel inhibitor nifedipine. The ryanodine receptor inhibitor dantrolene did not alter the initial Ca(++) influx but partially reduced the peak of Ca(++) i. These data suggest that Meth enhanced Ca(++) influx through membrane Ca(++) channels, which then triggered the release of Ca(++) from the endoplasmic reticulum in the cytosol. AAV-GCaMP5 was also injected to the parietal cortex of adult rats. Administration of Meth enhanced fluorescence in the ipsilateral cortex. Using immunohistochemistry, Meth-induced green fluorescence was found in the NeuN-containing cells in the cortex, suggesting that Meth increased Ca(++) in neurons in vivo. In conclusion, we have used in vitro and in vivo techniques to demonstrate a rapid increase of Ca(++) i by Meth in cortical neurons through overexpression of GCaMP5. As Meth induces behavioral responses and neurotoxicity through Ca(++) i, modulation of Ca(++) i may be useful to reduce Meth-related reactions.

Methamphetamine (MA) is a drug of abuse as well as a dopaminergic neurotoxin. 9-Cis retinoic acid (9cRA), a biologically active derivative of vitamin A, has protective effects against damage caused by H(2)O(2) and oxygen-glucose deprivation in vitro as well as infarction and terminal deoxynucleotidyl transferase-mediated dNTP nick-end labeling (TUNEL) labeling in ischemic brain. The purpose of this study was to examine if there was a protective role for 9cRA against MA toxicity in nigrostriatal dopaminergic neurons. Primary dopaminergic neurons, prepared from rat embryonic ventral mesencephalic tissue, were treated with MA. High doses of MA decreased tyrosine hydroxylase (TH) immunoreactivity while increasing TUNEL labeling. These toxicities were significantly reduced by 9cRA. 9cRA also inhibited the export of Nur77 from nucleus to cytosol, a response that activates apoptosis. The interaction of 9cRA and MA in vivo was next examined in adult rats. 9cRA was delivered intracerebroventricularly; MA was given (5 mg/kg, 4×) one day later. Locomotor behavior was measured 2 days after surgery for a period of 48 h. High doses of MA significantly reduced locomotor activity and TH immunoreactivity in striatum. Administration of 9cRA antagonized these changes. Previous studies have shown that 9cRA can induce bone morphogenetic protein-7 (BMP7) expression and that administration of BMP7 attenuates MA toxicity. We demonstrated that MA treatment significantly reduced BMP7 mRNA expression in nigra. Noggin (a BMP antagonist) antagonized 9cRA-induced behavioral recovery and 9cRA-induced normalization of striatal TH levels. Our data suggest that 9cRA has a protective effect against MA-mediated neurodegeneration in dopaminergic neurons via upregulation of BMP.

Proglucagon-derived peptides, especially glucagon-like peptide-1 (GLP-1) and its long-acting mimetics, have exhibited neuroprotective effects in animal models of stroke. Several of these peptides are in clinical trials for stroke. Oxyntomodulin (OXM) is a proglucagon-derived peptide that co-activates the GLP-1 receptor (GLP-1R) and the glucagon receptor (GCGR). The neuroprotective action of OXM, however, has not been thoroughly investigated. In this study, the neuroprotective effect of OXM was first examined in human neuroblastoma (SH-SY5Y) cells and rat primary cortical neurons. GLP-1R and GCGR antagonists, and inhibitors of various signaling pathways were used in cell culture to characterize the mechanisms of action of OXM. To evaluate translation in vivo, OXM-mediated neuroprotection was assessed in a 60-minute, transient middle cerebral artery occlusion (MCAo) rat model of stroke. We found that OXM dose- and time-dependently increased cell viability and protected cells from glutamate toxicity and oxidative stress. These neuroprotective actions of OXM were mainly mediated through the GLP-1R. OXM induced intracellular cAMP production and activated cAMP-response element-binding protein (CREB). Furthermore, inhibition of the PKA and MAPK pathways, but not inhibition of the PI3K pathway, significantly attenuated the OXM neuroprotective actions. Intracerebroventricular administration of OXM significantly reduced cerebral infarct size and improved locomotor activities in MCAo stroke rats. Therefore, we conclude that OXM is neuroprotective against ischemic brain injury. The mechanisms of action involve induction of intracellular cAMP, activation of PKA and MAPK pathways and phosphorylation of CREB.

Recent studies have demonstrated that the human placenta is a novel source of adult stem cells. We have provided laboratory evidence that transplantation of these human placenta-derived cells in vitro and in vivo stroke models promotes functional recovery. However, the mechanisms underlying these observed therapeutic benefits of human placenta-derived cells unfortunately remain poorly understood. Here, we examined the expression of two discrete types of melatonin receptors and their roles in proliferation and differentiation of cultured human amniotic epithelial cells (AECs). Cultured AECs express melatonin receptor type 1A (MT1), but not melatonin receptor type 1B (MT2). The proliferation of cultured AECs was increased in the melatonin-treated group in a dose-dependent manner, and the viability of cultured AECs could be further enhanced by melatonin. Moreover, the viability of AECs significantly decreased with H(2) O(2) exposure, which was reversed by pretreatment with melatonin, resulting in increased cell survival rate and cell proliferation. Immunocytochemically, administration of melatonin significantly suppressed nestin proliferation, but enhanced TUJ1 differentiation of MT1-expressing AECs. Additional experiments incorporating antibody blocking and synergistic AEC-melatonin treatments further showed AEC therapeutic benefits via MT1 modulation. Finally, analysis of trophic factors revealed cultured AECs secreted VEGF in the presence of melatonin. These data indicate that melatonin by stimulating MT1 increased cell proliferation and survival rate while enhancing neuronal differentiation of cultured AECs, which together with VEGF upregulation, rendered neuroprotection against experimental in vitro models of ischemic and oxidative stress injury.

The therapeutic goal in treating cerebral ischemia is to reduce the extent of brain injury and thus minimize neuro-logical impairment. We examined the effects of p-hydro-xybenzyl alcohol (HBA), an active component of Gastrodia elata Blume, on transient focal cerebral ischemia-induced brain injury with respect to the involvement of protein disulphide isomerase (PDI), nuclear factor-E2-related factor 2 (Nrf2), and neurotrophic factors. All animals were ovariectomized 14 days before ischemic injury. Ischemic injury was induced for 1 h by middle cerebral artery occlusion (MCAO) followed by 24-h reperfusion. Three days before MCAO, the vehicle-treated and the HBA-treated groups received intramuscular sesame oil and HBA (25 mg/kg BW), respectively. 2,3,5-Triphenyltetrazolium chloride (TTC) staining showed decreased infarct volume in the ischemic lesion of HBA-treated animals. HBA pretreatment also promoted functional recovery, as measured by the modified neurological severity score (mNSS; p < 0.05). Moreover, expression of PDI, Nrf2, BDNF, GDNF, and MBP genes increased by HBA treatment. In vitro, H2O2-induced PC12 cell death was prevented by 24 h HBA treatment, but bacitracin, a PDI inhibitor, attenuated this cytoprotective effect in a dose-dependent manner. HBA treatment for 2 h also induced nuclear translocation of Nrf2, possibly activating the intracellular antioxidative system. These results suggest that HBA protects against brain damage by modulating cytoprotective genes, such as Nrf2 and PDI, and neurotrophic factors.

This study assessed the potential of intravenous transplantation of human umbilical cord blood (HUCB) CD34+ cells transfected with glial cell line-derived neurotrophic factor (GDNF) gene to exert therapeutic benefits in spontaneous hypertensive rats (SHR) exposed to transient middle cerebral artery occlusion (MCAO). SHR with MCAO were randomly assigned to receive intravenously transplantation of vehicle, the plasmid containing the enhanced green fluorescent protein (pEGFP)-CD34+ cells or pEGFP-GDNF-CD34+ cells at 6h after stroke. The CD34+ cells transfected with GDNF gene expressed higher levels of GDNF mRNA and protein than nontransfected HUCB CD34+ cells in vitro. At 28 days after transplantation of GDNF gene modified CD34+ cells, significantly more GFP positive cells, neurons, and astrocytes, likely derived from the grafted cells, populated the peri-infarct area compared to those injected with pEGFP-CD34+ cells or vehicle. Furthermore, the stroke animals transplanted with GDNF gene modified CD34+ cells showed a significant increase in GDNF level in the infarcted hemisphere, reduced brain infarction volume, and enhanced functional recovery compared with those that received pEGFP-CD34+ cells. This study supports the use of a combined gene and stem cell therapy for treating stroke.

Cell therapy remains an experimental treatment for neurological disorders. A major obstacle in pursuing the clinical application of this therapy is finding the optimal cell type that will allow benefit to a large patient population with minimal complications. A cell type that is a complete match of the transplant recipient appears as an optimal scenario. Here, we report that menstrual blood may be an important source of autologous stem cells. Immunocytochemical assays of cultured menstrual blood reveal that they express embryonic-like stem cell phenotypic markers (Oct4, SSEA, Nanog), and when grown in appropriate conditioned media, express neuronal phenotypic markers (Nestin, MAP2). In order to test the therapeutic potential of these cells, we used the in vitro stroke model of oxygen glucose deprivation (OGD) and found that OGD-exposed primary rat neurons that were co-cultured with menstrual blood-derived stem cells or exposed to the media collected from cultured menstrual blood exhibited significantly reduced cell death. Trophic factors, such as VEGF, BDNF, and NT-3, were up-regulated in the media of OGD-exposed cultured menstrual blood-derived stem cells. Transplantation of menstrual blood-derived stem cells, either intracerebrally or intravenously and without immunosuppression, after experimentally induced ischemic stroke in adult rats also significantly reduced behavioral and histological impairments compared to vehicle-infused rats. Menstrual blood-derived cells exemplify a source of “individually tailored” donor cells that completely match the transplant recipient, at least in women. The present neurostructural and behavioral benefits afforded by transplanted menstrual blood-derived cells support their use as a stem cell source for cell therapy in stroke.

We recently demonstrated that blood–brain barrier permeabilization using mannitol enhances the therapeutic efficacy of systemically administered human umbilical cord blood (HUCB) by facilitating the entry of neurotrophic factors from the periphery into the adult stroke brain. Here, we examined whether the same blood–brain barrier manipulation approach increases the therapeutic effects of intravenously delivered HUCB in a neonatal hypoxic-ischaemic (HI) injury model. Seven-day-old Sprague–Dawley rats were subjected to unilateral HI injury and then at day 7 after the insult, animals intravenously received vehicle alone, mannitol alone, HUCB cells (15k mononuclear fraction) alone or a combination of mannitol and HUCB cells. Behavioural tests at post-transplantation days 7 and 14 showed that HI animals that received HUCB cells alone or when combined with mannitol were significantly less impaired in motor asymmetry and motor coordination compared with those that received vehicle alone or mannitol alone. Brain tissues from a separate animal cohort from the four treatment conditions were processed for enzyme-linked immunosorbent assay at day 3 post-transplantation, and revealed elevated levels of GDNF, NGF and BDNF in those that received HUCB cells alone or when combined with mannitol compared with those that received vehicle or mannitol alone, with the combined HUCB cells and mannitol exhibiting the most robust neurotropic factor up-regulation. Histological assays revealed only sporadic detection of HUCB cells, suggesting that the trophic factor–mediated mechanism, rather than cell replacement per se, principally contributed to the behavioural improvement. These findings extend the utility of blood–brain barrier permeabilization in facilitating cell therapy for treating neonatal HI injury.

Increasing evidence have supported that Wharton's jelly mesenchymal stem cell (WJ-MSCs) have immunomodulatory and protective effects against several diseases including kidney, liver pathologies, and heart injury. Few in vitro studies have reported that WJ-MSCs reduced inflammation in hippocampal slices after oxygen–glucose deprivation. We recently reported the neuroprotective effects of human WJ-MSCs (hWJ-MSCs) in rats exposed to a transient right middle cerebral artery occlusion. hWJ-MSCs transplantation significantly reduced brain infarction and microglia activation in the penumbra leading with a significant reduction of neurological deficits. Interestingly, the grafted hWJ-MSCs in the ischemic core were mostly incorporated into IBA1 (+) cells, suggesting that hWJ-MSCs were immunorejected by the host. The immune rejection of hWJ-MSCs was reduced in after cyclosporine A treatment. Moreover, the glia cell line-derived neurotrophic factor expression was significantly increased in the host brain after hWJ-MSCs transplantation. In conclusion, these results suggest that the protective effect of hWJ-MSCs may be due to the secretion of trophic factors rather than to the survival of grafted cells. This paper is a review article. Referred literature in this paper has been listed in the references section. The data sets supporting the conclusions of this article are available online by searching various databases, including PubMed. Some original points in this article come from the laboratory practice in our research center and the authors’ experiences.

Recent laboratory evidence implicates matrix metalloproteinases (MMPs) as playing a pivotal role in ischemic and traumatic brain injuries (TBI). Here, quantitative real-time PCR analyses revealed that brains from TBI rats displayed significantly elevated MMP-9 expression at 24 h post-TBI, which remained upregulated at least until 48 h after injury. Immunohistochemical analyses similarly revealed significantly increased MMP-9 immunoreactivity at 24 and 48 h post-TBI. These results demonstrate that alterations in MMPs (i.e., MMP-9) commenced immediately after TBI, suggesting that treatment strategies designed to maintain MMP integrity should be initiated in the acute phase of injury.

This study evaluated the effects of estrogen on the neuronal differentiation of human umbilical cord blood mesenchymal stem cells. Human umbilical cord blood mesenchymal stem cells cultured in a neuronal differentiation medium containing dimethylsulfoxide and butylated hydroxyanisole showed the expression of the neuronal cell-specific protein marker, beta-tubulin III. The estrogen treatment increased the proportion of neurons and neurite branching but reduced the mean neurite length. The relative expression of neurotropic factors such as brain-derived neurotropic factor, glial cell derived neurotropic factor, nerve growth factor, neurotrophin-3, and growth-associated protein 43 were higher in the estrogen-treated group than in the nontreated and estrogen receptor antagonist (ICI-182,780)-treated groups. These results suggest that estrogen stimulates the differentiation of neurons derived from human umbilical cord blood mesenchymal stem cells through the gene expression of neurotrophic factors.