Lesional accumulation of CD8<Superscript>+</Superscript> cells in sciatic nerves of experimental autoimmune neuritis rats

Experimental autoimmune neuritis (EAN) is a well-known animal model of human demyelinating polyneuropathies. Macrophages are the major immune cells in peripheral nerves and may exert tissue-damage or tissue-protective activity during EAN. While considered to define a subpopulation of T lymphocytes, CD8 expression has been found on certain macrophages that show cytotoxic effects. Here we have studied the spatiotemporal accumulation of CD8⁺ cells in sciatic nerves of EAN rats. A robust accumulation of CD8⁺ cells was observed in the sciatic nerves of EAN rats, which was positively correlated with the severity of neurological signs in EAN. Moreover, double-labelling experiments showed that the major cellular sources of CD8 were reactive macrophages. Therefore, our data here suggest a pathological role of CD8⁺ macrophages in EAN, which makes CD8⁺ macrophage a potential therapeutic target for EAN.     

Monocytes are essential for the neuroprotective effect of human cord blood cells following middle cerebral artery occlusion in rat

Systemic administration of human umbilical cord blood (HUCB) mononuclear cells (MNC) following middle cerebral artery occlusion (MCAO) in the rat reduces infarct size and, more importantly, restores motor function. The HUCB cell preparation is composed of immature T-cells, B-cells, monocytes and stem cells. In this study we examined whether the beneficial effects of HUCB injection were attributable to one of these cell types. Male Sprague Dawley rats underwent permanent MCAO followed 48 h later by intravenous administration of HUCB MNC preparations depleted of either CD14 ⁺ monocytes, CD133 ⁺ stem cells, CD2 ⁺ T-cells or CD19 ⁺ B cells. Motor function was measured prior to MCAO and 30 days post-stroke. When CD14 ⁺ monocytes were depleted from the HUCB MNC, activity and motor asymmetry were similar to the MCAO only treated animals. Monocyte depletion prevented HUCB cell treatment from reducing infarct size while monocyte enrichment was sufficient to reduce infarct size. Administration of monocyte-depleted HUCB cells did not suppress Iba1 labeling of microglia in the infarcted area relative to treatment with the whole HUCB preparation. These data demonstrate that the HUCB monocytes provide the majority of the efficacy in reducing infarct volume and promoting functional recovery.     

Dexamethasone suppresses infiltration of RhoA<Superscript>+</Superscript> cells into early lesions of rat traumatic brain injury

Inflammatory cell infiltration is a major part of secondary tissue damage in traumatic brain injury (TBI). RhoA is an important member of Rho GTPases and is involved in leukocyte migration. Inhibition of RhoA and its downstream target, Rho-associated coiled kinase (ROCK), has been proven to promote axon regeneration and function recovery following injury in the central nervous system (CNS). Previously, we showed that dexamethasone, an immunosuppressive corticosteroid, attenuated early expression of three molecules associated with microglia/macrophages activation following TBI in rats. Here, the effects of dexamethasone on the early expression of RhoA have been investigated in brains of TBI rats by immunohistochemistry. In brains of rats treated with TBI alone, significant RhoA⁺ cell accumulation was observed at 18 h post-injury and continuously increased during our observed time period. The accumulated RhoA⁺ cells were distributed to the areas of pannecrosis and selective neuronal loss. Most accumulated RhoA⁺ cells were identified as active microglia/macrophages by double-labelling. Dexamethasone (1 mg/kg body weight) was intraperitoneally injected on day 0 and 2 immediately following brain injury. Numbers of RhoA⁺ cells were significantly reduced on day 1 and 2 following administration of dexamethasone but returned to vehicle control level on day 4. However, dexamethasone treatment did not change the proportion of RhoA⁺ cells. These observations suggest that dexamethasone has only a transient effect on early leukocyte recruitment.     

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

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

Changes in Binding of [<Superscript>123</Superscript>I]CLINDE,a High-Affinity Translocator Protein 18 kDa (TSPO) Selective Radioligand in a Rat Model of Traumatic Brain Injury

After traumatic brain injury (TBI), secondary injuries develop, including neuroinflammatory processes that contribute to long-lasting impairments. These secondary injuries represent potential targets for treatment and diagnostics. The translocator protein 18 kDa (TSPO) is expressed in activated microglia cells and upregulated in response to brain injury and therefore a potential biomarker of the neuroinflammatory processes. Second-generation radioligands of TSPO, such as [¹²³I]CLINDE, have a higher signal-to-noise ratio as the prototype ligand PK11195. [¹²³I]CLINDE has been employed in human studies using single-photon emission computed tomography to image the neuroinflammatory response after stroke. In this study, we used the same tracer in a rat model of TBI to determine changes in TSPO expression. Adult Sprague–Dawley rats were subjected to moderate controlled cortical impact injury and sacrificed at 6, 24, 72 h and 28 days post surgery. TSPO expression was assessed in brain sections employing [¹²³I]CLINDE in vitro autoradiography. From 24 h to 28 days post surgery, injured animals exhibited a marked and time-dependent increase in [¹²³I]CLINDE binding in the ipsilateral motor, somatosensory and parietal cortex, as well as in the hippocampus and thalamus. Interestingly, binding was also significantly elevated in the contralateral M1 motor cortex following TBI. Craniotomy without TBI caused a less marked increase in [¹²³I]CLINDE binding, restricted to the ipsilateral hemisphere. Radioligand binding was consistent with an increase in TSPO mRNA expression and CD11b immunoreactivity at the contusion site. This study demonstrates the applicability of [¹²³I]CLINDE for detailed regional and quantitative assessment of glial activity in experimental models of TBI.     

Engraftment of freshly isolated or cultured human umbilical cord blood cells and the effect of cyclosporin A on the outcome

Human umbilical cord blood (HUCB) is a potentially valuable resource for cell therapy. The present study investigated the short-term survival of intrastriatal grafts of either freshly isolated or cultured HUCB cells and the effect of the immunosuppressive agent cyclosporin A (CSA) in host rat brains. The group injected with either freshly isolated or cultured HUCB cells was subdivided into CSA or saline controls. Freshly isolated and cultured HUCB cells displayed surface markers CD33, CD44, CD45, CD51/61 and CD90/Thy-1. The hematopoietic progenitor marker CD34 was expressed only in freshly isolated cells. The majority of injected HUCB cells were localized within a 500-mum radius from the injection site in the striatum; however, a subpopulation migrated along the corpus callosum. There was no significant statistical difference in the cell count between freshly isolated and cultured HUCB cells with or without CSA. Some grafted HUCB cells expressed either a neural or microglial marker. There was weak up-regulation of major histocompatibility complex (MHC) class I antigen in rats either with or without CSA. However, there were considerably fewer positive cells labeled with an MHC class II antigen in CSA groups. These results suggest that neither freshly isolated nor cultured HUCB cells induce acute rejection after intrastriatal transplantation up to 14 days. CSA suppressed up-regulation of MHC class II antigen in the host brain.     

Antiviral CD8<Superscript>+</Superscript> T cells cause an experimental autoimmune encephalomyelitis-like disease in naive mice

Major histocompatibility complex class I-restricted CD8⁺ cytotoxic T lymphocytes are involved in the pathogenesis of multiple sclerosis (MS) and both autoimmune, experimental autoimmune encephalomyelitis, and viral, Theiler’s murine encephalomyelitis virus (TMEV) infection, animal models of MS. Following TMEV infection, certain T cell hybridomas, generated from cloned TMEV-induced CD8⁺ T cells, were able to produce clinical signs of disease (flaccid hind limb paralysis) upon adoptive transfer into naive mice. Dual T cell receptors (TCR) are present on the surface of these cells as both Vβ3 and Vβ6 were detected by polymerase chain reaction (PCR) screening and flow cytometry and multiple Vα mRNAs were detected by PCR screening. This is the first demonstration of antiviral CD8⁺ T cells having more than one TCR initiating an autoimmune disease in the natural host of the virus. We hypothesize that this is a potential mechanism for virus-induced autoimmune disease initiated by CD8⁺ T cells.     

CXCR4+CD45− BMMNC subpopulation is superior to unfractionated BMMNCs for protection after ischemic stroke in mice

Cell-based therapy is considered to be a promising therapeutic strategy for stroke treatment. Although unfractionated bone marrow mononuclear cells (BMMNCs) have been tried in both preclinical and clinical trials, the effective subpopulations need to be identified. In this study, we used fluorescence-activated cell sorting to harvest the CXCR4⁺CD45⁺ and CXCR4⁺CD45⁻ BMMNC subpopulations from transgenic mice that express enhanced green fluorescent protein. We then allogeneically grafted unfractionated BMMNCs or a subpopulation into mice subjected to transient middle cerebral artery occlusion (tMCAO) and compared the effects on stroke outcomes. We found that CXCR4⁺CD45⁻ BMMNCs, but not CXCR4⁺CD45⁺ BMMNCs, more effectively reduced infarction volume and neurologic deficits than did unfractionated BMMNCs. Brain tissue from the ischemic hemisphere of mice treated with CXCR4⁺CD45⁻ BMMNCs had higher levels of vascular endothelial growth factor and lower levels of TNF-α than did tissue from mice treated with unfractionated BMMNCs. In contrast, CXCR4⁺CD45⁺ BMMNCs showed an increase in TNF-α. Additionally, CXCR4⁺CD45⁺ and CXCR4⁺CD45⁻ populations exhibited more robust migration into the lesion areas and were better able to express cell-specific markers of different linages than were the unfractionated BMMNCs. Endothelial and astrocyte cell markers did not colocalize with eGFP⁺ cells in the brains of tMCAO mice that received CXCR4⁺CD45⁺ BMMNCs. In vitro, the CXCR4⁺CD45⁻ BMMNCs expressed significantly more Oct-4 and Nanog mRNA than did the unfractionated BMMNCs. However, we did not detect gene expression of these two pluripotent markers in CXCR4⁺CD45⁺ BMMNCs. Taken together, our study shows for the first time that the CXCR4⁺CD45⁻ BMMNC subpopulation is superior to unfractionated BMMNCs in ameliorating cerebral damage in a mouse model of tMCAO and could represent a new therapeutic approach for stroke treatment.     

Lack of NG2 exacerbates neurological outcome and modulates glial responses after traumatic brain injury

Traumatic brain injury (TBI) is a major cause of death and disability. The underlying pathophysiology is characterized by secondary processes including neuronal death and gliosis. To elucidate the role of the NG2 proteoglycan we investigated the response of NG2‐knockout mice (NG2‐KO) to TBI. Seven days after TBI behavioral analysis, brain damage volumetry and assessment of blood brain barrier integrity demonstrated an exacerbated response of NG2‐KO compared to wild‐type (WT) mice. Reactive astrocytes and expression of the reactive astrocyte and neurotoxicity marker Lcn2 (Lipocalin‐2) were increased in the perilesional brain tissue of NG2‐KO mice. In addition, microglia/macrophages with activated morphology were increased in number and mRNA expression of the M2 marker Arg1 (Arginase 1) was enhanced in NG2‐KO mice. While TBI‐induced expression of pro‐inflammatory cytokine genes was unchanged between genotypes, PCR array screening revealed a marked TBI‐induced up‐regulation of the C‐X‐C motif chemokine 13 gene Cxcl13 in NG2‐KO mice. CXCL13, known to attract immune cells to the inflamed brain, was expressed by activated perilesional microglia/macrophages seven days after TBI. Thirty days after TBI, NG2‐KO mice still exhibited more pronounced neurological deficits than WT mice, up‐regulation of Cxcl13, enhanced CD45+ leukocyte infiltration and a relative increase of activated Iba‐1+/CD45+ microglia/macrophages. Our study demonstrates that lack of NG2 exacerbates the neurological outcome after TBI and associates with abnormal activation of astrocytes, microglia/macrophages and increased leukocyte recruitment to the injured brain. These findings suggest that NG2 may counteract neurological deficits and adverse glial responses in TBI. GLIA 2016;64:507–523     

CD8<Superscript>+</Superscript> T cells patrol HSV-1-infected trigeminal ganglia and prevent viral reactivation

A hallmark of herpes viruses is their capacity to cause recurrent disease. Recurrences of herpes simplex virus (HSV)-1 disease do not result from reinfection from external sources, but rather from reactivation of virus that is maintained in a latent state in sensory neurons and periodically reactivates from latency to cause recurrent disease. Recent findings implicate HSV-specific CD8⁺ T cells in immune surveillance of HSV-1 latently infected sensory neurons in trigeminal ganglia (TG) and inhibition of HSV-1 reactivation from latency. This review summarizes recent findings regarding the characteristics of the TG-resident CD8⁺ T cell population and certain unique obstacles that might complicate the development of therapeutic vaccines.     

Na<Superscript>+</Superscript>,K<Superscript>+</Superscript>-ATPase activity in an animal model of mania

We evaluated Na⁺,K⁺-ATPase activity in hippocampus of rats submitted to an animal model of mania which included the use of lithium and valproate. In the acute treatment, amphetamine or saline was administered to rats for 14 days, between day 8 and 14, rats were treated with lithium, valproate or saline. In the maintenance treatment, rats were treated with lithium, valproate or saline, between day 8 and 14, amphetamine or saline were administered. Locomotor activity was assessed by open field test and Na⁺,K⁺-ATPase activity was measured. Our results showed that mood stabilizers reversed and prevented amphetamine-induced behavioral effects. Moreover, amphetamine (acute treatment) increased Na⁺,K⁺-ATPase activity, and administration of lithium or valproate reversed this effect. In the maintenance treatment, amphetamine increased Na⁺,K⁺-ATPase activity in saline-pretreated rats. Amphetamine administration in lithium- or valproate-pretreated animals did not alter Na⁺,K⁺-ATPase activity. The findings suggest that amphetamine-induced hyperactivity may be associated with an increase in Na⁺,K⁺-ATPase.     

Methylphenidate treatment increases Na<Superscript>+</Superscript>, K<Superscript>+</Superscript>-ATPase activity in the cerebrum of young and adult rats

Methylphenidate is a central nervous system stimulant used for the treatment of attention-deficit hyperactivity disorder. Na⁺, K⁺-ATPase is a membrane-bound enzyme necessary to maintain neuronal excitability. Considering that methylphenidate effects on central nervous system metabolism are poorly known and that Na⁺, K⁺-ATPase is essential to normal brain function, the purpose of this study was to evaluate the effect of this drug on Na⁺, K⁺-ATPase activity in the cerebrum of young and adult rats. For acute administration, a single injection of methylphenidate (1.0, 2.0, or 10.0 mg/Kg) or saline was given to rats on postnatal day 25 or postnatal day 60, in the young and adult groups, respectively. For chronic administration, methylphenidate (1.0, 2.0, or 10.0 mg/Kg) or saline injections were given to young rats starting at postnatal day 25 once daily for 28 days. In adult rats, the same regimen was performed starting at postnatal day 60. Our results showed that acute methylphenidate administration increased Na⁺, K⁺-ATPase activity in hippocampus, prefrontal cortex, and striatum of young and adult rats. In young rats, chronic administration of methylphenidate also enhanced Na⁺, K⁺-ATPase activity in hippocampus and prefrontal cortex, but not in striatum. When tested in adult rats, Na⁺, K⁺-ATPase activity was increased in all cerebral structures studied. The present findings suggest that increased Na⁺, K⁺-ATPase activity may be associated with neuronal excitability caused by methylphenidate.     

Severe Hyperhomocysteinemia Decreases Respiratory Enzyme and Na<Superscript>+</Superscript>-K<Superscript>+</Superscript> ATPase Activities,and Leads to Mitochondrial Alterations in Rat Amygdala

Severe hyperhomocysteinemia is caused by increased plasma levels of homocysteine (Hcy), a methionine derivative, and is associated with cerebral disorders. Creatine supplementation has emerged as an adjuvant to protect against neurodegenerative diseases, due to its potential antioxidant role. Here, we examined the effects of severe hyperhomocysteinemia on brain metabolism, and evaluated a possible neuroprotective role of creatine in hyperhomocysteinemia, by concomitant treatment with Hcy and creatine (50 mg/Kg body weight). Hyperhomocysteinemia was induced in young rats (6-day-old) by treatment with homocysteine (0.3–0.6 µmol/g body weight) for 23 days, and then the following parameters of rat amygdala were evaluated: (1) the activity of the respiratory chain complexes succinate dehydrogenase, complex II and cytochrome c oxidase; (2) mitochondrial mass and membrane potential; (3) the levels of necrosis and apoptosis; and (4) the activity and immunocontent of Na⁺,K⁺-ATPase. Hcy treatment decreased the activities of succinate dehydrogenase and cytochrome c oxidase, but did not alter complex II activity. Hcy treatment also increased the number of cells with high mitochondrial mass, high mitochondrial membrane potential, and in late apoptosis. Importantly, creatine administration prevented some of the key effects of Hcy administration on the amygdala. We also observed a decrease in the activity and immunocontent of the α1 subunit of the Na⁺,K⁺-ATPase in amygdala after Hcy- treatment. Our findings support the notion that Hcy modulates mitochondrial function and bioenergetics in the brain, as well as Na⁺,K⁺-ATPase activity, and suggest that creatine might represent an effective adjuvant to protect against the effects of high Hcy plasma levels.     

CD11c‐positive cells from brain,spleen, lung,and liver exhibit site‐specific immune phenotypes and plastically adapt to new environments

The brain's immune privilege has been also attributed to the lack of dendritic cells (DC) within its parenchyma and the adjacent meninges, an assumption, which implies maintenance of antigens rather than their presentation in lymphoid organs. Using mice transcribing the green fluorescent protein under the promoter of the DC marker CD11c (itgax), we identified a juxtavascular population of cells expressing this DC marker and demonstrated their origin from bone marrow and local microglia. We now phenotypically compared this population with CD11c/CD45 double‐positive cells from lung, liver, and spleen in healthy mice using seven‐color flow cytometry. We identified unique, site‐specific expression patterns of F4/80, CD80, CD86, CX3CR1, CCR2, FLT3, CD103, and MHC‐II. Furthermore, we observed the two known CD45‐positive populations (CD45^high and CD45^int) in the brain, whereas liver, lung, and spleen exhibited a homogeneous CD45^high population. CD11c‐positive microglia lacked MHC‐II expression and CD45^high/CD11c‐positive cells from the brain have a lower percentage of MHC‐II‐positive cells. To test whether phenotypical differences are fixed by origin or specifically develop due to environmental factors, we transplanted brain and spleen mononuclear cells on organotypic slice cultures from brain (OHSC) and spleen (OSSC). We demonstrate that adaption and ramification of MHC‐II‐positive splenocytes is paralleled by down‐regulation of MHC‐II, whereas brain‐derived mononuclear cells neither ramified nor up‐regulated MHC‐II in OSSCs. Thus, brain‐derived mononuclear cells maintain their MHC‐II‐negative phenotype within the environment of an immune organ. Intraparenchymal CD11c‐positive cells share immunophenotypical characteristics of DCs from other organs but remain unique for their low MHC‐II expression. GLIA 2015;63:611–625     

Differential regulation of the monocytic calcium-binding peptides macrophage-inhibiting factor related protein-8 (MRP8/S100A8) and allograft inflammatory factor-1 (AIF-1) following human traumatic brain injury

Intracellular calcium (Ca²⁺) has been shown to function as second messenger and to be associated with activation of different cell types including microglia. Previously, in human focal cerebral infarctions an early expression of macrophage-related protein-8 (MRP8/ S100A8), a member of the Ca²⁺-binding S100-protein family, in microglia has been reported. On the other hand, a delayed activation of microglia was observed following traumatic brain injury (TBI). We therefore examined immunohistochemically microglial expression of MRP8 and allograft inflammatory factor-1 (AIF-1), identical to microglial response factor-1 (mrf-1) and ionized calcium binding adaptor molecule-1 (iba1) in human brains after TBI and in control brains. Both, MRP8 and AIF-1 are Ca²⁺-binding peptides which have been associated with microglial activation in experimental models and in human cerebral infarctions. Detection of AIF-1 in controls confirmed constitutive expression of this peptide in a subset of microglial cells. After TBI, the density of AIF-1⁺ microglia did not increase significantly. Lesional expression of AIF-1 did not significantly differ from other brain regions. Furthermore, following TBI, we found no significant differences in the density of AIF-1⁺ microglia as compared to controls. Microglial MRP8 expression was not detectable in controls and within the first 3 days post TBI, but increased rapidly after 3 days post TBI, suggesting a subpopulation of microglial cells to be AIF-1^–/MRP8⁺. We conclude that the delayed expression of MRP8 and the lack of AIF-1 up-regulation in microglia after TBI is in contrast to ischemic brain lesions and might reflect different activation cascades of microglia. Received: 24 January 2000 / Revised, accepted: 24 February 2000     

Bone marrow stromal cells and bone marrow‐derived mononuclear cells: Which are suitable as cell source of transplantation for mice infarct brain?

There are few studies that denote whether bone marrow stromal cells (BMSC) and bone marrow‐derived mononuclear cells (MNC) show the same therapeutic effects, when directly transplanted into the infarct brain. This study therefore aimed to compare their biological properties and behaviors in the infarct brain. Mouse BMSC were harvested and cultured. Mouse MNC were obtained through centrifugation techniques. Their cell markers were analyzed with FACS analysis. The MNC (10⁶ cells; n = 10) or BMSC (2 × 10⁵ cells; n = 10) were stereotactically transplanted into the ipsilateral striatum of the mice subjected to permanent middle cerebral artery occlusion at 7 days after the insult. Their survival, migration, and differentiation in the infarct brain were precisely analyzed using immunohistochemistry 4 weeks after transplantation. The MNC were positive for CD34, CD45, CD90, but were negative for Sca‐1. The BMSC were positive for CD90 and Sca‐1. The transplanted BMSC, but not MNC, extensively migrated into the peri‐infarct area. Approximately 20% of the transplanted BMSC expressed a neuronal marker, NeuN in the infarct brain, although only 1.4% of the transplanted MNC expressed NeuN. These findings strongly suggest that there are large, biological differences between MNC and BMSC as cell sources of regenerative medicine for ischemic stroke.