Positive Cell Densities Research Articles

Low intensity repetitive transcranial magnetic stimulation modulates brain-wide functional connectivity to promote anti-correlated c-Fos expression

Repetitive transcranial magnetic stimulation (rTMS) induces action potentials to induce plastic changes in the brain with increasing evidence for the therapeutic importance of brain-wide functional network effects of rTMS; however, the influence of sub-action potential threshold (low-intensity; LI-) rTMS on neuronal activity is largely unknown. We investigated whether LI-rTMS modulates neuronal activity and functional connectivity and also specifically assessed modulation of parvalbumin interneuron activity. We conducted a brain-wide analysis of c-Fos, a marker for neuronal activity, in mice that received LI-rTMS to visual cortex. Mice received single or multiple sessions of excitatory 10 Hz LI-rTMS with custom rodent coils or were sham controls. We assessed changes to c-Fos positive cell densities and c-Fos/parvalbumin co-expression. Peak c-Fos expression corresponded with activity during rTMS. We also assessed functional connectivity changes using brain-wide c-Fos-based network analysis. LI-rTMS modulated c-Fos expression in cortical and subcortical regions. c-Fos density changes were most prevalent with acute stimulation, however chronic stimulation decreased parvalbumin interneuron activity, most prominently in the amygdala and striatum. LI-rTMS also increased anti-correlated functional connectivity, with the most prominent effects also in the amygdala and striatum following chronic stimulation. LI-rTMS induces changes in c-Fos expression that suggest modulation of neuronal activity and functional connectivity throughout the brain. Our results suggest that LI-rTMS promotes anticorrelated functional connectivity, possibly due to decreased parvalbumin interneuron activation induced by chronic stimulation. These changes may underpin therapeutic rTMS effects, therefore modulation of subcortical activity supports rTMS for treatment of disorders involving subcortical dysregulation.

Inhaled Carbon Dioxide Improves Neurological Outcomes by Downregulating Hippocampal Autophagy and Apoptosis in an Asphyxia-Induced Cardiac Arrest and Resuscitation Rat Model.

Background Protracted cerebral hypoperfusion following cardiac arrest (CA) may cause poor neurological recovery. We hypothesized that inhaled carbon dioxide (CO2) could augment cerebral blood flow (CBF) and improve post-CA neurological outcomes. Methods and Results After 6-minute asphyxia-induced CA and resuscitation, Wistar rats were randomly allocated to 4 groups (n=25/group) and administered with different inhaled CO2 concentrations, including control (0% CO2), 4% CO2, 8% CO2, and 12% CO2. Invasive monitoring was maintained for 120 minutes, and neurological outcomes were evaluated with neurological function score at 24 hours post-CA. After the 120-minute experiment, CBF was 242.3% (median; interquartile range, 221.1%-267.4%) of baseline in the 12% CO2 group while CBF fell to 45.8% (interquartile range, 41.2%-58.1%) of baseline in the control group (P<0.001). CBF increased along with increasing inhaled CO2 concentrations with significant linear trends (P<0.001). At 24 hours post-CA, compared with the control group (neurological function score, 9 [interquartile range, 8-9]), neurological recovery was significantly better in the 12% CO2 group (neurological function score, 10 [interquartile range, 9.8-10]) (P<0.001) while no survival difference was observed. Brain tissue malondialdehyde (P=0.02) and serum neuron-specific enolase (P=0.002) and S100β levels (P=0.002) were significantly lower in the 12% CO2 group. TUNEL (terminal deoxynucleotidyl transferase-mediated biotin-deoxyuridine triphosphate nick-end labeling)-positive cell densities in hippocampal CA1 (P<0.001) and CA3 (P<0.001) regions were also significantly reduced in the 12% CO2 group. Western blotting showed that beclin-1 (P=0.02), p62 (P=0.02), and LAMP2 (lysosome-associated membrane protein 2) (P=0.01) expression levels, and the LC3B-II:LC3B-I ratio (P=0.02) were significantly lower in the 12% CO2 group. Conclusions Administering inhaled CO2 augmented post-CA CBF, mitigated oxidative brain injuries, ameliorated neuronal injury, and downregulated apoptosis and autophagy, thereby improving neurological outcomes.

Task-Related c-Fos Expression in the Posterior Parietal Cortex During the "Rubber Tail Task" Is Diminished in Ca2+-Dependent Activator Protein for Secretion 2 (Caps2)-Knockout Mice.

Rubber hand illusion (RHI), a kind of body ownership illusion, is sometimes atypical in individuals with autism spectrum disorder; however, the brain regions associated with the illusion are still unclear. We previously reported that mice responded as if their own tails were being touched when rubber tails were grasped following synchronous stroking to rubber tails and their tails (a “rubber tail illusion”, RTI), which is a task based on the human RHI; furthermore, we reported that the RTI response was diminished in Ca2+-dependent activator protein for secretion 2-knockout (Caps2-KO) mice that exhibit autistic-like phenotypes. Importance of the posterior parietal cortex in the formation of illusory perception has previously been reported in human imaging studies. However, the local neural circuits and cell properties associated with this process are not clear. Therefore, we aimed to elucidate the neural basis of the RTI response and its impairment by investigating the c-Fos expression in both wild-type (WT) and Caps2-KO mice during the task since the c-Fos expression occurred soon after the neural activation. Immediately following the delivery of the synchronous stroking to both rubber tails and actual tails, the mice were perfused. Subsequently, whole brains were cryo-sectioned, and each section was immunostained with anti-c-Fos antibody; finally, c-Fos positive cell densities among the groups were compared. The c-Fos expression in the posterior parietal cortex was significantly lower in the Caps2-KO mice than in the WT mice. Additionally, we compared the c-Fos expression in the WT mice between synchronous and asynchronous conditions and found that the c-Fos-positive cell densities were significantly higher in the claustrum and primary somatosensory cortex of the WT mice exposed to the synchronous condition than those exposed to the asynchronous condition. Hence, the results suggest that decreased c-Fos expression in the posterior parietal cortex may be related to impaired multisensory integrations in Caps2-KO mice.

AB0125 CLINICAL CHARACTERISTICS OF RHEUMATOID ARTHRITIS PATIENTS WITH IGG4-RELATED SYNOVITIS

Background:Elevated serum IgG4 (sIgG4) and IgG4+ plasma cell tissue infiltration are outstanding features of IgG4-related disease (IgG4-RD). However, elevated IgG4 is not specific for IgG4-RD. Our previous study reported elevated sIgG4 in 46% of rheumatoid arthritis (RA) patients (Mediators Inflamm 2014). Whether synovium from RA patients show similar characteristics of IgG4-RD and how about the clinical characteristics of RA patients with IgG4-related synovitis have not been reported yet.Objectives:To explore the serum and synovial IgG4 level and their correlation with disease indicators in RA.Methods:Active RA patients who underwent needle synovial biopsy with qualified synovium tissue were recruited. Demographic and clinical data were collected simultaneously. Synovium tissue were stained with H&amp;E for Krenn synovitis score and immunohistochemistry for positive cell densities of CD20, CD38, IgG and IgG4. Serum IgG4 level was detected by immunonephelometry.Results:Among 96 RA patients recruited, 74 (77.1%) were female, the median age was 55.0 (46.0~61.0) years, disease duration was 42.0 (12.0~120.0) months and SDAI was 31.2 (22.1~42.8).The median sIgG4 was 1.38 (0.86~2.42) g/L and 49(51.0%) patients had elevated sIgG4. Compared with those with normal sIgG4, RA patients with elevated sIgG4 had significantly higher levels of PrGA [7 (5~8) vs. 6 (4~7)], ESR [90 (64~116) mm/h vs. 61 (38~75) mm/h], CRP [46.20 (17.20~74.20) mg/L vs. 18.90 (9.46~49.20) mg/L], DAS28-ESR [6.3 (5.6~7.4) vs. 5.7 (4.7~6.4)], SDAI [34.2 (25.3~48.8) vs. 27.8 (18.9~35.9)] and HAQ-DI [1.70 (0.61~2.28) vs. 0.88 (0.40~1.75), all P&lt;0.05]. Meanwhile, they also showed significantly higher synovial counts of CD38+ plasma cells [1240(559~2290) /mm2 vs. 1020(354~1777) /mm2], IgG4+ plasma cells [106 (39~249) /mm2 vs. 68 (3~123) /mm2], and higher ratio of IgG4+/IgG+ plasma cells [26.3 (15.5~38.0) % vs. 15.2 (0.9~24.7) %, all P&lt;0.05].The median IgG4+ plasma cells count was 83 (10~192) /mm2 and median ratio of IgG4+/IgG+ plasma cells was 19.1 (8.4~31.5)%. Both of them correlated positively with ESR, CRP and sIgG4 (r=0.216~0.394, all P&lt;0.05). There were 46 (47.9%) patients with IgG4+ plasma cells &gt;10/HPF, who had significant higher ESR [86 (50~109) mm/h vs. 65 (40~84) mm/h] and CRP [43.35 (16.93~77.85) mg/L vs. 26.15 (9.54~52.53) mg/L, both P&lt;0.05] than those with IgG4+ plasma cells ≤10/HPF. There were 13 (13.5%) patients with the ratio of IgG4+/IgG+ plasma cells &gt;40%, and 11 (11.5%) patients with both IgG4+ plasma cells &gt;10/HPF and IgG4+/IgG+ plasma cells ratio &gt;40% (IgG4-related synovitis). RA patients with IgG4-related synovitis had significant higher ESR than the others [106 (53~125) mm/h vs. 69 (41~91) mm/h, P&lt;0.05].There were 10 (10.4%) patients showing elevated sIgG4 and IgG4-related synovitis. Four patients completed 1-year follow-up and all of them achieved remission at 6th month (SDAI≤3.3, Figure 1). Only one patient had radiographic progression at 12th month.Figure 1.Dynamic disease activity of 4 RA patients with elevated sIgG4 and IgG4-related synovitis during 1-year follow-up.Conclusion:IgG4-related synovitis can be found in RA patients. Their clinical significance in disease characteristics and outcomes are worth further study.Acknowledgements:This work was supported by National Natural Science Foundation of China (no. 81971527, 81801606 and 81801605), Guangdong Natural Science Foundation (no. 2018A030313541 and 2018A030313690), Guangdong Medical Scientific Research Foundation (no. A2018062), Guangdong Basic and Applied Basic Research Foundation (no. 2019A1515011928 and 2020A1515110061), and Science and Technology Program of Guangzhou (no. 201904010088).Disclosure of Interests:None declared

Abstract 3857: Characterization of immune cell biomarkers in undifferentiated pleomorphic sarcoma

Abstract Background: Undifferentiated pleomorphic sarcoma (UPS) are genetically complex sarcomas often showing a relatively higher immune infiltration among sarcomas, and a demonstrated benefit for immunotherapy in a subset of patients. This study characterizes the immune microenvironment of UPS including tumor infiltrating lymphocytes (TILs), as well as immune checkpoint (IC) and related biomarkers. Methods: We used FFPE surgical resected UPS from 90 patients (43 primary, 28 recurrence and 19 metastasis) placed in a tissue microarray and stained with immunohistochemistry for the following biomarkers: IC (stimulatory: OX40 &amp; ICOS; inhibitory: PD-L1, LAG3, IDO1, &amp; PD1), TILs (CD3 &amp; CD8), monocytes-macrophages (CD163), Adenosine Pathway (CD73 &amp; CD39), and pSTAT3. We scored TIL and IC biomarkers as positive cell densities, and PD-L1, pSTAT3, CD73 and CD39 as percentage of expression in malignant cells. Clinicopathological data and clinical outcome were available for all patients. Spearman correlation was used to assess the correlation between two biomarkers. Cox proportional hazard regression models were used to identify the prognostic biomarkers for overall survival (OS), recurrence-free survival (RFS), and metastasis-free survival (MFS). Results: In our cohort, PD-L1, pSTAT3, CD73 and CD39 positive expression (cut-off ≥1%) was 19, 73 78 and 68% respectively. These biomarkers were positively correlated with immune infiltrates: CD3 (p=0.013, 0.009, 0.009, &amp;lt;.001), CD8 (p=0.004, 0.016, &amp;lt;.001, &amp;lt;.001) and ICOS (p=0.005, 0.222, 0.007, 0.022). CD73 and CD39 were also positively correlated with both CD163 (p=0.001, &amp;lt;.001) and PD-1 (p=0.012, &amp;lt;.001). In recurrent tumors, smaller tumors had higher CD3, CD8, ICOS and PD-1 (p=0.011, 0.045, 0.013, 0.045). In metastatic tumor, CD163 and OX40 were positively correlated with age (p=0.045, 0.019). OX40 was inversely correlated with tumor size (p=0.04). ICOS positively correlated with PD-1, PD-L1 and CD39 (p&amp;lt;.001, 0.002, 0.003) in primary tumors; with IDO1 and PD1 (p=0.012, &amp;lt;.001) in recurrent tumors; and with PD-L1 expression (p=0.031) in metastatic tumors. Low CD3, CD8, CD163 densities (cut-off median) were associated with inferior OS (HR: 3.19 [1.37, 7.42]; p=0.007), RFS (HR: 2.79 [1.21, 6.42], p=0.016), and MFS (HR: 2.42 [1.05, 5.56]; p=0.038) after adjusting for size, respectively. Low CD73 and PD-L1 (cut-off median) were associated with inferior OS (HR 3.03 [HR: 1.21, 7.61], p=0.018), RFS (HR: 2.59 [1.06, 6.36]; p=0.037), and MFS (HR: 2.43 [1.00, 5.89], p=0.049), respectively. Conclusions: In this study, we characterized the immune cell infiltrates and the expression of PD-L1, pSTAT3, CD73 and CD39 in UPS. These biomarkers differentially correlated with clinicopathological characteristics in primary, recurrent and metastatic tumors. Lower TILs and low CD73/PD-L1 expression were associated with inferior survival outcomes. Citation Format: Carmelia M. Barreto, Luisa M. Solis-Soto, Ruth Salazar, Swati Gite, Edwin R. Parra, Barbara Mino, Davis Ingram, Khalida M. Wani, Cheuk H. Leung, Heather Lin, Ignacio I. Wistuba, Alexander Lazar, Wei-Lien Wang. Characterization of immune cell biomarkers in undifferentiated pleomorphic sarcoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3857.

Object and food novelty induce distinct patterns of c-fos immunoreactivity in amygdala and striatum in domestic male chicks (Gallus gallus domesticus)

Avoidance of novelty, termed neophobia, protects animals from potential dangers but can also impair their adaptation to novel environments or food resources. This behaviour is particularly well described in birds but the neurobiological correlates remain unexplored. Here, we measured neuronal activity in the amygdala and the striatum, two brain regions believed to be involved in novelty detection, by labelling the early gene c-fos following chicks exposure to a novel food (NF), a novel object (NO) or a familiar food (FF). NF and NO chicks showed significantly longer latencies to touch the food, less time eating and emitted more fear-vocalizations than control chicks. Latency to touch the food was also longer for NO than for NF chicks. Significantly higher densities of c-fos positive cells were present in all the nuclei of the arcopallium/amygdala of NF and NO chicks compared to FF chicks. Also, NO chicks showed higher positive cell densities than NF chicks in the posterior amygdaloid, the intermediate and the medial arcopallium. Exposure to novel food or object induced a similar increase in c-fos expression in the nucleus accumbens and the medial striatum. Our data provide evidence activation of the arcopallium/amygdala is specific of the type of novelty. The activation of striatum may be more related to novelty seeking.

Environmental Enrichment From Birth Impacts Parvalbumin Expressing Cells and Wisteria Floribunda Agglutinin Labelled Peri-Neuronal Nets Within the Developing Murine Striatum.

Environmental enrichment can dramatically affect both the development and function of neural circuits. This is accomplished, at least in part, by the regulation of inhibitory cellular networks and related extracellular matrix glycoprotein structures known as perineuronal nets. The degree to which enhanced housing can influence brain areas involved in the planning and execution of actions is not well known. We examined the effect of enriching mice from birth on parvalbumin expression and perineuronal net formation in developing and adult striatum. This input nucleus of the basal ganglia consists of topographically discernible regions that serve different functions, providing a means of simultaneously examining the influence of environmental factors on discrete, but related networks. Greater densities of striatal parvalbumin positive cells and wisteria floribunda agglutinin labelled perineuronal nets were present in enriched pups during the second postnatal week, primarily within the lateral portion of the nucleus. Housing conditions continued to have an impact into adulthood, with enriched mice exhibiting higher parvalbumin positive cell densities in both medial and lateral striatum. Curiously, no differences due to housing conditions were detected in striatal perineuronal net densities of mature animals. The degree of overlap between striatal parvalbumin expression and perineuronal net formation was also increased, suggesting that heightened neural activity associated with enrichment may have contributed to greater engagement of networks affiliated with cells that express the calcium binding protein. Brain derived neurotrophic factor, an important regulator of inhibitory network maturation, is also subtly, but significantly affected within the striatum of enriched cohorts. Together, these findings suggest that environmental enrichment can exert cell specific effects within different divisions of an area vital for the regulation of action.

The Combined Extract of Black Sticky Rice and Dill Improves Poststroke Cognitive Impairment in Metabolic Syndrome Condition.

Despite the increase in cognitive deficit following stroke in metabolic syndrome (MetS) condition, the therapeutic strategy is still limited. Since oxidative stress and neuroinflammation play the crucial roles on the pathophysiology of aforementioned conditions, the cognitive enhancing effect of the combined extract of Oryza sativa and Anethum graveolens was considered based on their antioxidant, anti-inflammation, and neuroprotective effects together with the synergistic effect concept. Male Wistar rats weighing 180-220 g were induced metabolic syndrome-like condition by using a high-carbohydrate high-fat diet (HCHF diet). Then, reperfusion injury following cerebral ischemia was induced by the occlusion of right middle cerebral artery and treated with the combined extract of O. sativa and A. graveolens (OA extract) at doses of 0.5, 5, and 50 mg/kg BW once daily for 21 days. Spatial memory was assessed every 7 days throughout the experimental period. At the end of the study, neuron and glial fibrillary acidic protein- (GFAP-) positive cell densities, the oxidative stress status, AChE, and the expression of proinflammatory cytokines (TNF-α, IL-6) in the hippocampus were determined. The results showed that OA extract at all doses used in this study significantly improved memory together with the reductions of MDA, TNF-α, IL-6, AChE, and density of GFAP-positive cell but increased neuron density in the hippocampus. Taken together, OA is the potential cognitive enhancer in memory impairment following stroke in MetS condition. The possible underlying mechanism may occur partly via the reductions of oxidative stress status, GFAP-positive cell density, and neuroinflammatory cytokines such as TNF-α and IL-6 together with the suppression of AChE activity in the hippocampus. This study suggests that OA is the potential functional ingredient to improve the cognitive enhancer. However, further clinical research is required.

Therapeutic Effects of Umbilical Cord Blood-Derived Mesenchymal Stem Cells Combined with Polydeoxyribonucleotides on Full-Thickness Rotator Cuff Tendon Tear in a Rabbit Model

While therapies using mesenchymal stem cells (MSCs) to treat rotator cuff tendon tear (RCTT) have yielded some promising preliminary results, MSCs therapy has not yet completely regenerated full-thickness RCTT (FTRCTT). It has recently been reported that polydeoxyribonucleotide (PDRN) is effective in the treatment of chronic rotator cuff disease. We hypothesized that local injection of human umbilical cord blood-derived (UCB)–MSCs with PDRN would be more effective in regenerating tendon tear than UCB-MSCs alone. The purpose of this study was to evaluate the effects of UCB–MSCs combined with different doses of PDRN on the regeneration of RCTT in a chronic RCTT model by using a rabbit model. New Zealand white rabbits (n = 24) with FTRCTT were allocated randomly into three groups (8 rabbits per group). Three different injectants (G1-S, 0.2 mL UCB-MSCs; G2-P1, 0.2 mL UCB-MSCs with one injection of 0.2 mL PDRN; G3-P4, 0.2 mL UCB-MSCs, and four injections of 0.2 mL PDRN per week) were injected into FTRCTT under US-guidance. After the rabbits were euthanized, we evaluated ross morphological and histological change. Motion analysis was also performed. There were significant differences in gross morphological changes between before, and at 4 weeks after injection, in all three groups, but no differences were found among the three groups. Masson’s trichrome (MT) or anti-type 1 collagen antibody (COL-1)-positive cell densities in G2-P1 and G3-P4 were improved significantly compared with those in G1-S, but showed no significant difference between G2-P1 and G3-P4. On motion analysis, walking distance and fast walking time in G2-P1 and G3-P4 were significantly longer/higher than those in G1-S, but showed no significant differences between G2-P1 and G3-P4. These results demonstrated that there was no significant difference in the gross morphologic change of tendon tear between UCB-MSCs only and combination with PDRN injection in rabbit model of chronic traumatic FTRCTT. Furthermore, there were no significant differences in the regenerative effects between high and low doses of (0.8 and 0.2) mL of PDRN.

Immunopathological Features of Severe Chronic Atopic Keratoconjunctivitis and Effects of Topical Cyclosporine Treatment

ABSTRACTPurpose: To assess differential roles of inflammatory cells in pathophysiology of severe atopic keratoconjunctivitis (AKC) and evaluate immunomodulatory effects of topical cyclosporine A (CsA).Methods: A total of 10 patients with severe, steroid-dependent/resistant chronic active AKC were treated using frequent topical CsA 0.05% as monotherapy for 2 months. Conjunctival biopsy specimens before and after treatment were examined using immunohistochemistry. A total of 10 healthy age-matched adults served as the control group.Results: Baseline AKC samples revealed greater cluster of differentiation 4 (CD4), interferon gamma (IFNγ), human leukocyte antigen–D-related (HLA-DR) positive cell densities compared with healthy controls (P < 0.05), as well as interleukin (IL)-17 (P = 0.08). Topical CsA treatment induced a significant reduction in CD4 and IL-17 expressions (P < 0.05); post-treatment levels were same as normals (P > 0.05). Despite reduction after treatment (P = 0.06), HLA-DR expression remained higher than controls (P < 0.05).Conclusions: AKC-related conjunctival inflammation appears to be mediated by delayed hypersensitivity. In this short-term trial, frequent topical CsA improved conjunctival inflammation.

Developmental Ethanol-Induced Sleep Fragmentation, Behavioral Hyperactivity, Cognitive Impairment and Parvalbumin Cell Loss are Prevented by Lithium Co-treatment

Developmental ethanol exposure is a well-known cause of lifelong cognitive deficits, behavioral hyperactivity, emotional dysregulation, and more. In healthy adults, sleep is thought to have a critical involvement in each of these processes. Our previous work has demonstrated that some aspects of cognitive impairment in adult mice exposed at postnatal day 7 (P7) to ethanol (EtOH) correlate with slow-wave sleep (SWS) fragmentation (Wilson et al., 2016). We and others have also previously demonstrated that co-treatment with LiCl on the day of EtOH exposure prevents many of the anatomical and physiological impairments observed in adults. Here we explored cognitive function, diurnal rhythms (activity, temperature), SWS, and parvalbumin (PV) and perineuronal net (PNN)-positive cell densities in adult mice that had received a single day of EtOH exposure on P7 and saline-treated littermate controls. Half of the animals also received a LiCl injection on P7. The results suggest that developmental EtOH resulted in adult behavioral hyperactivity, cognitive impairment, and reduced SWS compared to saline controls. Both of these effects were reduced by LiCl treatment on the day of EtOH exposure. Finally, developmental EtOH resulted in decreased PV/PNN-expressing cells in retrosplenial (RS) cortex and dorsal CA3 hippocampus at P90. As with sleep and behavioral activity, LiCl treatment reduced this decrease in PV expression. Together, these results further clarify the long-lasting effects of developmental EtOH on adult behavior, physiology, and anatomy. Furthermore, they demonstrate the neuroprotective effects of LiCl co-treatment on this wide range of developmental EtOH’s long-lasting consequences.

Association between tumor necrosis factor – A gene -1031T/C promoter polymorphism and endometriosis

Developmental ethanol exposure is a well-known cause of lifelong cognitive deficits, behavioral hyperactivity, emotional dysregulation, and more. In healthy adults, sleep is thought to have a critical involvement in each of these processes. Our previous work has demonstrated that some aspects of cognitive impairment in adult mice exposed at postnatal day 7 (P7) to ethanol (EtOH) correlate with slow-wave sleep (SWS) fragmentation (Wilson et al., 2016). We and others have also previously demonstrated that co-treatment with LiCl on the day of EtOH exposure prevents many of the anatomical and physiological impairments observed in adults. Here we explored cognitive function, diurnal rhythms (activity, temperature), SWS, and parvalbumin (PV) and perineuronal net (PNN)-positive cell densities in adult mice that had received a single day of EtOH exposure on P7 and saline-treated littermate controls. Half of the animals also received a LiCl injection on P7. The results suggest that developmental EtOH resulted in adult behavioral hyperactivity, cognitive impairment, and reduced SWS compared to saline controls. Both of these effects were reduced by LiCl treatment on the day of EtOH exposure. Finally, developmental EtOH resulted in decreased PV/PNN-expressing cells in retrosplenial (RS) cortex and dorsal CA3 hippocampus at P90. As with sleep and behavioral activity, LiCl treatment reduced this decrease in PV expression. Together, these results further clarify the long-lasting effects of developmental EtOH on adult behavior, physiology, and anatomy. Furthermore, they demonstrate the neuroprotective effects of LiCl co-treatment on this wide range of developmental EtOH’s long-lasting consequences.

Lentivirus-mediated microRNA-124 gene-modified bone marrow mesenchymal stem cell transplantation promotes the repair of spinal cord injury in rats

Our study aims to explore the effects of lentivirus-mediated microRNA-124 (miR-124) gene-modified bone marrow mesenchymal stem cell (BMSC) transplantation on the repair of spinal cord injury (SCI) in rats. BMSCs were isolated from the bone marrow of rats. The target gene miR-124 was identified using a luciferase-reporter gene assay. Seventy-two rats were selected for construction of the SCI model, and the rats were randomly divided into the blank group, sham group, SCI group, negative control (NC) group, overexpressed miR-124 group and si-PDXK group. The mRNA expression of miR-124 and the mRNA and protein expression of pyridoxal kinase (PDXK) were detected by quantitative real-time polymerase chain reaction and western blotting. The locomotor capacity of the rats was evaluated using the Basso, Beattie and Bresnahan (BBB) scale. Brdu, neuron-specific enolase (NSE), neurofilament (NF) and microtubule-associated protein 2 (MAP2) were detected using immunohistochemistry. The expression levels of thyrotropin-releasing hormone (TRH), prostacyclin (PGI2) and gangliosides (GM) were measured using an enzyme-linked immunosorbent assay. PDXK was identified as the target gene of miR-124. The overexpressed miR-124 group exhibited higher miR-124 expression than the SCI, NC and si-PDXK groups. Compared with the SCI and NC groups, the PDXK expression was downregulated in the overexpressed miR-124 and si-PDXK groups, and the BBB scores were significantly increased 7, 21 and 35 days after transplantation. The double-labeled positive cell densities (Brdu+NSE/NF/MAP2) and the expression levels of TRH, PGI2 and GM in the overexpressed miR-124 group were significantly higher than those in the NC and SCI groups. These results indicated that miR-124 targeted PDXK to accelerate the differentiation of BMSCs into neurocytes and promote SCI repair.

Investigation on etiology of hepatic venous obstruction Budd-Chiari syndrome.

Budd-Chiari syndrome (BCS) is an uncommon clinical condition with a complex etiology. Pathogenesis of BCS is still poorly understood. We included hepatic veno-occlusive lesion tissues of 20 patients (patients group) with hepatic venous obstruction BCS and compared with 20 similar tissues with other etiologies (control group). Morphological changes in hepatic veno-occlusive lesion tissues and the positive expression of proliferating cell nuclear antigen (PCNA), C-myc, and P-53 were observed by the pathological examination (H&E staining) and immunohistochemistry assay. Our results showed that PCNA and C-myc positive cell densities were significantly higher in patient group than control group. P-53 positive cell density showed increasing trends in patients than control group. Moreover, we observed irregular hyperplasia in intimal tissue, fibrous connective tissue, and smooth muscle cell, accompanied by tissue degeneration (hyaloid degeneration and fibrinoid degeneration) and a large quantity of inflammatory cell infiltration. In conclusion, an overexpression of PCNA, C-myc, and a weak positive expression of P53 might launch the extremely irregular hepatic venous intimal hyperplasia, which is probably one of the etiologies of hepatic venous obstruction BCS.

Effects of black cohosh and estrogen on the hypothalamic nuclei of ovariectomized rats at different temperatures

Ethnopharmacological relevanceCimicifuga racemosa (L.) Nutt. (CR), known as black cohosh, has been used in Europe as a medicinal plant for more than a century and its roots have been widely used for the treatment of menopausal symptoms. Remifemin, the main ingredient in liquid or tablet medications prepared from isopropyl alcohol extracts of black cohosh rhizome, has also been evaluated in clinical studies. ObjectivesTo observe changes in the expression of the c-Fos protein in the hypothalamic nuclei of four groups of rats—sham-operated group (SHAM), ovariectomized (OVX) group, ovariectomized group treated with estrogen(OVX+E), and ovariectomized group treated with the isopropanol extract of Cimicifuga racemosa (OVX+ICR)—and to investigate the mechanisms of black cohosh and estrogen that take place in the hypothalamic nuclei of ovariectomized rats. MethodsFifty rats were assigned to each of the four groups and placed in incubators at 4°C, 10°C, 25°C, 33°C, or 38°C for 2h. They were then anesthetized, and their brains were removed after heart perfusion. c-Fos expression in the hypothalamic nuclei was evaluated using immunohistochemical methods. ResultsIn the median preoptic nucleus (MnPO), ventromedial preoptic nucleus (VMPO), and suprachiasmatic nucleus (SCh) of the SHAM group, in the anterior hypothalamic area (AH) and supraoptic nucleus (SO) of all four groups, and in the paraventricular nucleus (PVN) of the SHAM, OVX and OVX+E groups, the c-Fos-positive cell densities all changed in a similar manner: the cell density decreased when the temperature was less than 25°C and the density increased when the temperature was greater than 25°C, demonstrating a V-type curve. The c-Fos density was lowest at 25°C. The other nuclei demonstrated irregular changes. The positive cell densities in the MnPO, AH, and PVN of the SHAM, OVX+E, and OVX+ICR groups were greater than the densities measured in the OVX group at all temperatures, except 25°C. Positive cell densities in the SHAM, OVX+E, and OVX+ICR groups were greater than the densities measured in the OVX groups in the MPA at 25°C, in the VMPO at 4°C, 33°C, and 38°C, in the SO at 4°C, 10°C, and 38°C, and in the SCh at 33°C. ConclusionRegardless of the temperature, positive cell densities were lower in the MnPO, MPA, VMPO, AH, SCh, SO, and PVN of the OVX groups in comparison with the densities measured in the same sites in the SHAM group. Following the administration of black cohosh and estrogen, the positive cell densities in the OVX groups increased and became closer to, or exceeded, those measured in the SHAM group, suggesting that both drugs may act on the hypothalamic nuclei and have therapeutic effects on menopausal symptoms.

Expression of NogoA in 21a area of visual cortex in visual developing normal cat and strabismic amblyopia cat

Background The study of the molecular mechanism of visual plasticity is helpful for the explaining and prevention of strabismus and amblyopia.The effect and significance of NogoA in the strabismus and amblyopia formation are attracting more attention. Objective The present study was to investigate the expression of NogoA in 21a area of visual cortex in strabismic-induced amblyopia cats and explore the possible molecular mechanism of strabismic-induced amblyopia. Methods Sixteen 4-week old clean cats were randomizedly divided into normal group and strabismic-induced amblyopia group and eight for each group.The strabismic-induced amblyopia models were created by cutting off the external rectus in 8 cats.Pattern visual evoked potentials ( P-VEP ) were recorded 1 week after operation and compared with normal cats,and depression of amplitude and prolongation of implied time of P100 wave were as the successful criterion of model.The 200 ml paraformaldehyde was infused via heart to fax the brain under the deep anesthesia and then the cats were sacrificed and the brain cortex sections were prepared.The morphology of 21a zone of cat visual cortex was examined by haematoxylin and eosin staining,and the expressions of NogoA in 21a area of visual cortex were detected by immunohistochemistry with a monoclonal antiNogoA antibody.The use of the animals complied with the Regulations for the Administration of Affair Concerning Experimental Animals by State Science and Technology Commission. Results The implied time and amplitude ratio of VEP P100 wave were (98.10±7.07)ms and (0.83±0.14) in normal group,and those in strabismic-induced amblyopia group were (108.50±6.95 )ms and (0.35 ±0.09 ),showing significant differences between two groups (t=4.450,P=0.005 ; t =5.970,P =0.005 ).The numbers of neurons were similar in 21a area of visual cortex between the two groups,but the volume of the neurons was lessen in strabismic-induced amblyopia group.The positive cell densities for NogoA were ( 387.37±2.01 ) cells/mm2,( 354.58± 1.85 ) cells/mm2 and ( 289.68± 1.81 ) cells/mm2 in layer Ⅱ/Ⅲ,Ⅳ and V/V[ respectively in strabismic-induced amblyopia group,and those in normal group were ( 161.39± 1.98 ) cells/mm2,( 128.93 ± 1.26 ) cells/mm2 and ( 96.25 ± 1.49 ) cells/mm2,indicating a significant increase in model cats ( t =- 160.400,- 201.890,- 164.740,P =0.000 ). Conclusions NogoA may play an important role in the regulation of the sensitive developmental period of visual sense and its plasticity. Key words: Amblyopia; Visual cortex; Immunohistochemistry; NogoA

Relationship between miR-218 and CDK6 expression and their biological impact on glioma cell proliferation and apoptosis

To investigate the relationship between the expression of miR-218 and CDK6 in glioma cells, and their biological impacts on the tumor cell proliferation and apoptosis. Expression levels of miR-218 as well as CDK6 and Ki-67 proteins were analyzed in 60 cases of gliomas with various grades and 10 control brain tissue samples by tissue microarray, locked oligonucleotide probe in situ hybridization and immunohistochemistry. Glioblastoma multiform cell line (U87MG) was transfected with miR-218 mimics (mimics group) and a control sequence (control group), followed by qRT-PCR detection of miR-218 and immunocytochemical stain of CDK6 and Ki-67, respectively. Single cell gel electrophoresis was used to detect the presence of apoptotic cell. The miR-218 labeling indexes (LI) were statistically different (P<0.05) among all groups including control (22.45 +/- 0.59) and various glioma groups (grades I - II 4.00 +/- 1.07, grade III 1.87 +/- 1.06 and grade IV 0.94 +/- 0.78, respectively). The CDK6 LI of the four groups was 7.25 +/- 1.20, 16.71 +/- 0.80, 24.43 +/- 0.62 and 32.05 +/- 0.43, respectively. Significant differences existed between the control group and the glioma groups, and between grade IV and grades I - II glioma groups (P<0.01). Ki-67 positive cell densities of the above four groups (0.00 +/- 0.00, 9.30 +/- 3.48, 31.15 +/- 9.44 and 60.15 +/- 13.60) were significantly different from one and another (P<0.01). The expression of miR-218 negatively correlated with CDK-6 LI (r = -0.480, P<0. 01) and Ki-67 positive cell density (r = - 0.534, P<0.01), while the latter two positively correlated with each other (r = 0.530, P<0.01). U87MG transfection experiment showed that the miR-218 level of the mimics group was significantly higher than that of the control group (P<0.01). CDK6 and Ki-67 LI of the mimics group (14.74 +/- 1.19 and 30.88 +/- 3.31) were significantly lower than those of the control group (79.06 +/- 2.07 and 64.94 +/- 3.96, P<0.01), whilst its apoptotic index (AI) (68.44 +/- 7.05) was significantly higher than that of the control group (13.04 +/- 0.97, P<0.01). The expression level of miR-218 is an important reference indicator for the assessment of the grade of gliomas. An aberrant decrease of its expression may lead to an increase of the CDK6 expression and proliferative activity of giloma cells. Introducing exogenous miR-218 may effectively down-regulate the CDK6 expression, inhibit cell proliferation and induce apoptosis of malignant giloma cells. These findings imply that miR-218 may serve as a therapeutic agent against malignant glioma.

Combination of intracortically administered VEGF and environmental enrichment enhances brain protection in developing rats

Postnatal development of the visual cortex is modulated by experience, especially during the critical period. In rats, a stable neuronal population is only acquired after this relatively prolonged period. Vascular endothelial growth factor (VEGF) is the most important angiogenic factor and also has strong neuroprotective, neurotrophic and neurogenic properties. Similar effects have been described for rearing in enriched environments. Our aim is to investigate the vascular and neuronal effects of combining VEGF infusion and environmental enrichment on the visual cortex during the initial days of the critical period. Results showed that a small percentage of Cleaved Caspase-3 positive cells colocalized with neuronal markers. The lesion produced by the cannula implantation resulted in decreased vascular, neuronal and Caspase-3 positive cell densities. Rearing under enriched environment was unable to reverse these effects in any group, whereas VEGF infusion alone partially corrected those effects. A higher effectiveness was reached by combining both the procedures, the most effective combination being when enriched-environment rearing was introduced only after minipump implantation. In addition to the angiogenic effect of VEGF, applied strategies also had synergic neuroprotective effects, and the combination of the two strategies had more remarkable effects than those achieved by each strategy applied individually.

Reduced production of sulfated glycosaminoglycans occurs in Zambian children with kwashiorkor but not marasmus

Background:Kwashiorkor, a form of severe malnutrition with high mortality, is characterized by edema and systemic abnormalities. Although extremely common, its pathophysiology remains poorly understood, and its characteristic physical signs are unexplained.Objective: Because kwashiorkor can develop in protein-losing enteropathy, which is caused by a loss of enterocyte heparan sulfate proteoglycan (HSPG), and previous observations suggest abnormal sulfated glycosaminoglycan (GAG) metabolism, we examined whether intestinal GAG and HSPG are abnormal in children with kwashiorkor.Design: Duodenal biopsy samples collected from Zambian children with marasmus (n = 18), marasmic kwashiorkor (n = 8), and kwashiorkor (n = 15) were examined for expression of HSPG, GAGs, and immunologic markers and compared against reference samples from healthy UK control children. GAG and HSPG expression density and inflammatory cell populations were quantitated by computerized analysis.Results: The kwashiorkor group was less wasted and had a lower HIV incidence than did the other groups. All duodenal biopsy samples showed inflammation compared with the histologically uninflamed control samples. Biopsy samples from marasmic children had greater inflammation and greater CD3+ and HLA-DR (human leukocyte antigen DR)–positive cell densities than did samples from children with kwashiorkor. Expression of both HSPG and GAGs was similar between marasmic and well-nourished UK children but was markedly lower in children with kwashiorkor in both the epithelium and lamina propria. Although underglycosylated and undersulfated, epithelial syndecan-1 protein was normally expressed in kwashiorkor, which confirmed that abnormalities arise after core protein synthesis.Conclusions: Intestinal HSPG loss occurs in kwashiorkor, which may precipitate protein-losing enteropathy to cause edema. If occurring systemically, impaired HSPG expression could cause several previously unexplained features of kwashiorkor. We speculate that a genetic predisposition to reduced HSPG biosynthesis may offer a contrasting selective advantage, by both diminishing protein catabolism during transient undernutrition and protecting against specific infectious diseases.

Statistical parametric mapping of immunopositive cell density

We developed a new method for comparing immunopositive cell densities across groups of animals and creating statistical parametric maps on standardized sections. As an example, we compared Iba-1 (microglial marker) positive cell densities in rats with ( n = 6) and without ( n = 6) unilateral injection of 1-methyl-4-phenylpyridinium salt (MPP +). Immunopositive cells were automatically counted in each animal over a coronal section in the midbrain (bregma −5.9 mm) and a positive cell density map was created for each animal. After the positive cell density map was normalized to a template section from an atlas, positive cell densities of the two groups were compared in each pixel over the section and a statistical parameter ( p-value from t-test) was mapped on each pixel. We were able to detect significant increases of microglias in the side of MPP + injection not only in the substantia nigra pars compacta but also in adjacent white matter. We also applied the same analysis to tyrosine hydroxylase stained sections and detected significant decreases of dopamine neurons in the side of MPP + injection. The new method was proven to be useful for detecting significant changes of cell densities over the entire area of immunostained sections.