Expression of Root Genes in Arabidopsis Seedlings Grown by Standard and Improved Growing Methods.

Roots of Arabidopsis thaliana seedlings grown in the laboratory using the traditional plant-growing culture system (TPG) were covered to maintain them in darkness. This new method is based on a dark chamber and is named the improved plant-growing method (IPG). We measured the light conditions in dark chambers, and found that the highest light intensity was dramatically reduced deeper in the dark chamber. In the bottom and side parts of dark chambers, roots were almost completely shaded. Using the high-throughput RNA sequencing method on the whole RNA extraction from roots, we compared the global gene expression levels in roots of seedlings from these two conditions and identified 141 differently expressed genes (DEGs) between them. According to the KEGG (Kyoto Encyclopedia of Genes and Genomes) enrichment, the flavone and flavonol biosynthesis and flavonoid biosynthesis pathways were most affected among all annotated pathways. Surprisingly, no genes of known plant photoreceptors were identified as DEGs by this method. Considering that the light intensity was decreased in the IPG system, we collected four sections (1.5 cm for each) of Arabidopsis roots grown in TPG and IPG conditions, and the spatial-related differential gene expression levels of plant photoreceptors and polar auxin transporters, including CRY1, CRY2, PHYA, PHYB, PHOT1, PHOT2, and UVR8 were analyzed by qRT-PCR. Using these results, we generated a map of the spatial-related expression patterns of these genes under IPG and TPG conditions. The expression levels of light-related genes in roots is highly sensitive to illumination and it provides a background reference for selecting an improved culture method for laboratory-maintained Arabidopsis seedlings.

Chronic stress hinders sensory axon regeneration via impairing mitochondrial cristae and OXPHOS.

Spinal cord injury (SCI) often leads to physical limitations, persistent pain, and major lifestyle shifts, enhancing the likelihood of prolonged psychological stress and associated disorders such as anxiety and depression. The mechanisms linking stress with regeneration remain elusive, despite understanding the detrimental impact of chronic stress on SCI recovery. In this study, we investigated the effect of chronic stress on primary sensory axon regeneration using a preconditioning lesions mouse model. Our data revealed that chronic stress-induced mitochondrial cristae loss and a decrease in oxidative phosphorylation (OXPHOS) within primary sensory neurons, impeding central axon regrowth. Corticosterone, a stress hormone, emerged as a pivotal player in this process, affecting satellite glial cells by reducing Kir4.1 expression. This led to increased neuronal hyperactivity and reactive oxygen species levels, which, in turn, deformed mitochondrial cristae and impaired OXPHOS, crucial for axonal regeneration. Our study underscores the need to manage psychological stress in patients with SCI for effective sensory-motor rehabilitation.

Tissue factor-factor VIIa complex induces epithelial ovarian cancer cell invasion and metastasis through a monocytes-dependent mechanism.

OBJECTIVE: Tumor-associated macrophage infiltration and up-regulation of tissue factor-factor VII (TF-FVIIa) complex have been observed in the peritoneum and stroma of epithelial ovarian cancer (EOC). However, it is not clear how tumor-associated macrophage and TF-FVIIa complex promotes EOC invasion. In the present study, we aimed to determine the mechanism by which interaction of TF-FVIIa and monocytes (MOs) promotes EOC metastasis. METHOD: Matrigel invasion assay was used to analyze the potential of EOC metastasis. Enzyme-linked immunosorbent assay and real-time polymerase chain reaction were used to detect expressions of cytokines and chemokines. Fluorescence-activated cell sorting was used to count the percentage of CD14, CD68, and CD163 of MOs. RESULTS: We found that the TF-FVIIa complex caused dynamic changes in MOs cytokine and chemokine expression. CD14 and CD163 were also upregulated on MOs by TF-FVIIa. Epithelial ovarian cancer cells were cocultured with TF-FVIIa-stimulated MOs, demonstrating increased invasion potential. Interleukin 8 (IL-8) was proposed as the major chemoattractant mediating EOC invasion based on MOs messenger RNA and protein expression profiles. Anti-IL-8 monoclonal neutralizing antibody attenuated EOC cell invasion in a concentration-dependent manner, and tumor necrosis factor α from TF-FVIIa-stimulated MOs was observed to amplify IL-8 production. The following transcription factors in MOs were activated by TF-FVIIa and inhibited by the tissue factor pathway inhibitor: oncogenes HIF-1α, HIF-1ß, Oct I, Oct II, and Egr-1; inflammatory mediators c-Fos and c-Rel; and STAT family members STAT5A and STAT5B. CONCLUSIONS: Our study suggested that the interaction between the TF-FVIIa complex might play a role in mediating EOC invasion and metastasis depending on MOs mechanism.

Surgical intervention combined with weight-bearing walking training improves neurological recoveries in 320 patients with clinically complete spinal cord injury: a prospective self-controlled study.

Although a large number of trials in the SCI field have been conducted, few proven gains have been realized for patients. In the present study, we determined the efficacy of a novel combination treatment involving surgical intervention and long-term weight-bearing walking training in spinal cord injury (SCI) subjects clinically diagnosed as complete or American Spinal Injury Association Impairment Scale (AIS) Class A (AIS-A). A total of 320 clinically complete SCI subjects (271 male and 49 female), aged 16-60 years, received early (≤ 7 days, n = 201) or delayed (8-30 days, n = 119) surgical interventions to reduce intraspinal or intramedullary pressure. Fifteen days post-surgery, all subjects received a weight-bearing walking training with the "Kunming Locomotion Training Program (KLTP)" for a duration of 6 months. The neurological deficit and recovery were assessed using the AIS scale and a 10-point Kunming Locomotor Scale (KLS). We found that surgical intervention significantly improved AIS scores measured at 15 days post-surgery as compared to the pre-surgery baseline scores. Significant improvement of AIS scores was detected at 3 and 6 months and the KLS further showed significant improvements between all pair-wise comparisons of time points of 15 days, 3 or 6 months indicating continued improvement in walking scores during the 6-month period. In conclusion, combining surgical intervention within 1 month post-injury and weight-bearing locomotor training promoted continued and statistically significant neurological recoveries in subjects with clinically complete SCI, which generally shows little clinical recovery within the first year after injury and most are permanently disabled. This study was approved by the Science and Research Committee of Kunming General Hospital of PLA and Kunming Tongren Hospital, China and registered at ClinicalTrials.gov (Identifier: NCT04034108) on July 26, 2019.

Thrombin facilitates invasion of ovarian cancer along peritoneum by inducing monocyte differentiation toward tumor-associated macrophage-like cells.

Peritoneal metastasis is a distinct pathologic characteristic of advanced epithelial ovarian cancer (EOC), which is the most deadly disease of the female reproductive tract. The inflammatory environment of the peritoneum in EOC contains abundant macrophages, activated thrombin, and thrombin-associated receptors. However, little is known about the mechanism by which the thrombin-macrophages interaction contributes to tumor invasion and metastasis. We investigated the phenotype and cytokine/chemokine expression of thrombin-treated peripheral blood monocytes (MOs)/macrophages, it was found that the phenotype of MOs was altered toward a TAM-like macrophage CD163(high)IL-10(high)CCL18(high)IL-8(high) after thrombin stimulation. By Matrigel invasion assay, the conditioned medium of thrombin-stimulated MOs accelerated remarkable invasion of ES-2, SKOV3, and HO-8910, which was similar to invasive cell numbers of ascites stimuli (P < 0.05) and higher than MOs medium alone (P < 0.05). IL-8 was proposed as the major chemoattractant mediating EOC invasion based on MOs mRNA and protein expression profiling. It was observed that anti IL-8 monoclonal neutralizing antibody attenuated EOC cell invasion in a concentration-dependent manner. Increased transcriptional activation of NF-kappaB p50/p65 was identified in thrombin-treated MOs. This study provided insight the role of thrombin in the regulation of EOC peritoneal invasion via "educating" MOs.

The interaction of coagulation factor XII and monocyte/macrophages mediating peritoneal metastasis of epithelial ovarian cancer.

OBJECTIVES: Pathological studies have indicated that the peritoneum of epithelial ovarian cancer (EOC) patients exhibits characteristics of chronic inflammation like peritonitis. Abundant macrophage infiltration and increased expression of coagulation factor XII (FXII) have been observed in the peritoneum of EOC patients. The aim of this study is to determine how the interaction between FXII and monocyte/macrophages (MO/MAs) contributes to EOC cell invasion and metastasis of the peritoneum. METHODS: MO/MAs from the peripheral blood of healthy female donors and tumor-associated macrophages (TAMs) from EOC ascites were collected and cultured. We assessed phenotypes, cytokine/chemokine production, and phagocytic function of FXII-treated MO/MAs. The effects of the FXII-MO/MAs interaction on EOC cell invasion were determined by the Matrigel in vitro invasion assay. In addition, signaling pathway mediators were evaluated for their potential roles in MO/MA activation. RESULTS: MO/MAs exhibited M2-polarized phenotypes after FXII treatment, which was CD163(high)IL-10(high)CCL18(high)IL-8(high)CCR2(high)CXCR2(high). The phagocytic potential of MO/MAs was also upregulated. Matrigel results indicated that invasion of EOC cells was enhanced when exposed to conditioned medium from FXII-stimulated MO/MAs. Transcription factors found to be upregulated in FXII-stimulated MO/MAs included Fra-1, Fra-2, Fos-B in the AP-1 family, oncogenes HIF-1 and Oct, and STAT-5A in the STAT family. CONCLUSIONS: FXII may facilitate EOC cell metastasis by transforming MO/MAs toward tumor-associated macrophage-like cells.

Transplantation of bone marrow mesenchymal stem cells reduces lesion volume and induces axonal regrowth of injured spinal cord.

It has been demonstrated that transplantation of bone marrow mesenchymal stem cells (BMSCs) improves recovery of injured spinal cord in animal models. However, the mechanism of how BMSCs promote repair of injured spinal cord remains under investigation. The present study investigated the neural differentiation of BMSCs, the lesion volume and axonal regrowth of injured spinal cord after transplantation. Seven days after spinal cord injury, 3 x 10(5) BMSCs or PBS (control) was delivered into the injury epicenter of the spinal cord. At 8 weeks after spinal cord injury, transplantation of BMSCs reduced the volume of cavity and increased spared white matter as compared to the control. BMSCs did not express the cell marker of neurons, astrocytes and oligodendrocytes in injured spinal cord. Transmission electron microscopic examination displayed an increase in the number of axons in BMSC rats. The effect of BMSCs on growth of neuronal process was further investigated by using a coculture system. The length and the number of neurites from spinal neurons significantly increased when they cocultured with BMSCs. PCR and immunochemical analysis showed that BMSCs expressed brain-derived neurotrophic factor (BDNF) and glia cell line-derived neurotrophic factor (GDNF). These findings demonstrate that transplantation of BMSCs reduces lesion volume and promotes axonal regrowth of injured spinal cord.

GDNF-enhanced axonal regeneration and myelination following spinal cord injury is mediated by primary effects on neurons.

We previously demonstrated that coadministration of glial cell line-derived neurotrophic factor (GDNF) with grafts of Schwann cells (SCs) enhanced axonal regeneration and remyelination following spinal cord injury (SCI). However, the cellular target through which GDNF mediates such actions was unclear. Here, we report that GDNF enhanced both the number and caliber of regenerated axons in vivo and increased neurite outgrowth of dorsal root ganglion neurons (DRGN) in vitro, suggesting that GDNF has a direct effect on neurons. In SC-DRGN coculture, GDNF significantly increased the number of myelin sheaths produced by SCs. GDNF treatment had no effect on the proliferation of isolated SCs but enhanced the proliferation of SCs already in contact with axons. GDNF increased the expression of the 140 kDa neural cell adhesion molecule (NCAM) in isolated SCs but not their expression of the adhesion molecule L1 or the secretion of the neurotrophins NGF, NT3, or BDNF. Overall, these results support the hypothesis that GDNF-enhanced axonal regeneration and SC myelination is mediated mainly through a direct effect of GDNF on neurons. They also suggest that the combination of GDNF administration and SC transplantation may represent an effective strategy to promote axonal regeneration and myelin formation after injury in the spinal cord.

In Vivo Exon Replacement in the Mouse Atp7b Gene by the Cas9 System.

The application of CRISPR/Cas9 has opened a new era in gene therapy, making it possible to correct mutated genomes in vivo. Exon replacement can correct many mutations and has potential clinical value. In this study, we used a lentivirus-delivered transgene to obtain transgenic mice in which Cas9 and green fluorescent protein (GFP) were driven by the hTBG promoter and were specifically expressed in the liver. In Cas9-positive mice, only ∼11.6% of hepatocytes were GFP positive. The newborn Cas9-positive F1 mice were injected via the temporal vein with rAAV carrying a modified homologous replacement sequence for exon 8 of Atp7b and a pair of single-strand guide RNAs targeting the introns surrounding exon 8. When the Cas9-positive hepatocytes were sorted and analyzed by PCR and next-generation deep sequencing with different labels, ∼16.34 ± 4.02% to 19.37 ± 6.50% of the analyzed copies of exon 8 were replaced by the donor template in the genome of GFP-positive hepatocytes, that is, 1.81 ± 0.29% to 2.09 ± 0.54% replacement occurred in all liver genomes. However, when rAAV carrying a modified homologous replacement sequence was injected into the adult spCas9 mice, a double-cut deletion ratio of up to 99%, only about 1.10-1.13% of the exon 8 replacement rate was detected in Cas9-positive hepatocytes. This study is the first to achieve exon replacement via CRISPR/Cas9, which will benefit research on CRISPR/Cas9 technology for gene therapy.

Expression and regulation of major histocompatibility complex on neural stem cells and their lineages.

The expression of major histocompatibility complex (MHC) antigens on neural stem cells (NSCs) and their lineages is tightly related to the fate of these cells as grafts in allogenic transplantation. In this study, we observed that NSCs derived from embryonic rat forebrain expressed MHC class I and class II molecules at a low level, whereas the cells differentiated from NSCs, including neurons, astrocytes, and oligodendrocytes, lost their MHC expression. However, a proinflammatory factor, interferon-gamma (IFN-gamma), could induce and up-regulate the expression of MHC in both NSCs and their differentiated lineages in vitro. These results suggest that predifferentiating NSCs into lineage-limited cells prior to transplantation combined with controlling the local production of proinflammatory cytokines moderately may potentially benefit the survival of transplants.

Effects of autoimmunity on recovery of function in adult rats following spinal cord injury.

The central nervous system (CNS) is considered to be an immune-privileged site. For a long time, autoimmunity-induced inflammation has been viewed as an important mediator of secondary damage in the CNS following injury. However, other studies also suggest that autoimmunity is protective and beneficial. To investigate whether protective autoimmunity is present following spinal cord injury (SCI), we employed neonatally thymectomized (Tx) rats which contain few T lymphocytes in their peripheral blood, and passively immunized them with T lymphocytes activated by myelin basic protein (MBP) or spinal cord homogenate (SCH). Here we report that, among Tx, sham-Tx (sTx) and normal rats that received a contusive SCI, no significant histological and behavioral differences were found, suggesting that the endogenous T lymphocytes had no significant influence on the pathogenesis of secondary SCI. In rats passively immunized with MBP- or SCH-activated T cells (MBP-T or SCH-T, respectively), similar numbers of CD4(+) T cells were found to infiltrate into the injured spinal cords. However, only the MBP-T immunization showed neuroprotection, evidenced by the reduction of post-traumatic neuronal losses and improvement of functional recovery. These results collectively suggest that not all T lymphocytes against CNS antigens are neuroprotective and that a subpopulation of them, such as those of MBP-T cells, could be beneficial for SCI repair.

[The skewed usage of T cell receptor beta variable chain at the maternal-fetal interface of women with unexplained recurrent spontaneous abortion].

OBJECTIVE: To investigate T cell receptor (TCR) variable beta (BV) chain usage at the maternal-fetal interface and explore the relationship between the skewed TCR BV usage and unexplained recurrent spontaneous abortion (RSA). METHODS: Eighteen cases with unexplained RSA, together with matched 41 women with normal pregnancies in first trimester from Renji Hospital, Shanghai Jiao Tong University were studied. A high-resolution spectrum typing analysis of complementarity-determining region 3 (CDR3) was used to detect and compare the degree and frequency of TCR BV family expression in deciduas between RSA patients and normal controls. RESULTS: (1) The expression degree of BV19 (0.029 +/- 0.031 vs. 0.013 +/- 0.010, P = 0.038) in RSA group showed a higher usage, while BV5.2 (0.040 +/- 0.035 vs. 0.067 +/- 0.052, P = 0.046) showed a significantly lower usage when compared with normal controls. No significant difference in the expression of the other TCR BV families between RSA and controls were observed (P > 0.05). (2) TCR BV2, 3, 6, and 7 were the four most common BV families in deciduas of patients with RSA and normal controls, whose frequencies were all more than 50%. In RSA group, higher frequencies of BV15 (33.3% vs. 7.3%, P = 0.018), BV19 (38.9% vs. 14.6%, P = 0.049) and BV20 (33.3% vs. 7.3%, P = 0.018) were observed; meanuhile lower frequencies of BV4 (33.3% vs. 65.9%, P = 0.026) and BV7 (66.7% vs.92.7%, P = 0.018) distributions were observed. The other TCR BV families did not display significantly different freqencies of distribution (P > 0.05). CONCLUSIONS: It is suggested that a significant skewed TCR BV family occurs at the maternal-fetal interface in patients who undergo abortion. The specific skewed usages of TCR BV might be associated with the susceptibility to unexplained pregnancy loss.

Neuroprotective effect of baicalin on focal cerebral ischemia in rats.

Baicalin, a flavonoid compound from the root of the herb Scutellaria baicalensis Georgi, has been widely used to treat patients with inflammatory disease. The aim of this study was to assess the efficacy of baicalin in a rat model of focal cerebral ischemia. Adult male Sprague-Dawley rat models of cerebral artery occlusion were established and then randomly and equally divided into three groups: ischemia (cerebral ischemia and reperfusion), valproic acid (cerebral ischemia and reperfusion + three intraperitoneal injections of valproic acid; positive control), and baicalin (cerebral ischemia and reperfusion + intraperitoneal injection of baicalin for 21 days). Neurological deficits were assessed using the postural reflex test and forelimb placing test at 3, 7, 14, and 21 days after ischemia. Rat cerebral infarct volume was measured using 2,3,5-triphenyltetrazolium chloride (TTC) staining method. Pathological change of ischemic brain tissue was assessed using hematoxylin-eosin staining. In the baicalin group, rat neurological function was obviously improved, cerebral infarct volume was obviously reduced, and the pathological impairment of ischemic brain tissue was obviously alleviated compared to the ischemia group. Cerebral infarct volume was similar in the valproic acid and baicalin groups. These findings suggest that baicalin has a neuroprotective effect on cerebral ischemia.

Production of Wilson Disease Model Rabbits with Homology-Directed Precision Point Mutations in the ATP7B Gene Using the CRISPR/Cas9 System.

CRISPR/Cas9 has recently been developed as an efficient genome engineering tool. The rabbit is a suitable animal model for studies of metabolic diseases. In this study, we generated ATP7B site-directed point mutation rabbits to simulate a major mutation type in Asians (p. Arg778Leu) with Wilson disease (WD) by using the CRISPR/Cas9 system combined with single-strand DNA oligonucleotides (ssODNs). The efficiency of the precision point mutation was 52.94% when zygotes were injected 14 hours after HCG treatment and was significantly higher than that of zygotes injected 19 hours after HCG treatment (14.29%). The rabbits carrying the allele with mutant ATP7B died at approximately three months of age. Additionally, the copper content in the livers of rabbits at the onset of WD increased nine-fold, a level similar to the five-fold increase observed in humans with WD. Thus, the efficiency of precision point mutations increases when RNAs are injected into zygotes at earlier stages, and the ATP7B mutant rabbits are a potential model for human WD disease with applications in pathological analysis, clinical treatment and gene therapy research.

A novel method for obtaining highly enriched Schwann cell populations from mature monkey nerves based on in vitro pre­degeneration.

Schwann cells (SCs) are indispensable for cell therapy and tissue engineering of the peripheral nervous system. Easy access to activated, highly proliferative SCs is necessary for clinical applications. The present study developed a fast, efficient method for obtaining highly purified SCs from the peripheral nerve of a mature Rhesus monkey. The common peroneal nerves of 4­year­old Rhesus monkeys were harvested and subjected to in vitro pre­degeneration in a modified SC culture medium (SCCM). The nerve pieces were subsequently treated enzymatically to dissociate the cells and then cultured for 2 days in SCCM. Cultured cells were treated with purification medium containing Ara­C to assist in restricting the overgrowth of fibroblast­like cells, for 24 h. After another 24­h cultivation period, the cells were subsequently treated with a multiplex collagenase, which enabled SC detachment over fibroblast detachment, and thereby facilitated SC isolation. Finally, SCs were cultured in SCCM. The cell yield was determined by cell counting following enzyme digestion and SC purity was determined from the percentage of SCs with respect to the total number of cells. Following purification, 96.3±3.9% of cells were identified as SCs. In vitro pre­degeneration in the presence of basic­fibroblast growth factor, heregulin ß1 and forskolin maximized the purity and yield of SCs that could be obtained from monkey peroneal nerves. The present study identified a novel technique that can efficiently isolate and purify SCs from mature monkey nerves based on in vitro pre­degeneration.

Expression of CD28 and cytotoxic T lymphocyte antigen 4 at the maternal-fetal interface in women with unexplained pregnancy loss.

OBJECTIVE: To investigate the expression of CD28 and CTLA-4, two costimulatory molecules involved in T-cell activation at the maternal-fetal interface in women with unexplained pregnancy loss. METHODS: A total of 57 women, 39 with unexplained spontaneous abortion (SA) and 18 with unexplained recurrent spontaneous abortion (RSA), were enrolled in the study. A high-resolution spectratyping analysis of fluorescent bands was performed to detect CD28 and CTLA-4 expression in decidual tissues. RESULTS: The mean expression of CTLA-4 mRNA was significantly higher in women with SA than in controls (P<0.05). Moreover, the expression of CTLA-4 was higher in SA patients with genotype AA and phenotype A+ (AA+AG) than in controls (P<0.01). The expression of CTLA-4 was not significantly different in patients with RSA and in controls. The expression of CD28 was significantly decreased in patients with RSA (P<0.01) and SA (P<0.05) compared with controls. The mean ratios of CTLA-4/CD28 were significantly higher in patients with RSA (P<0.01) and SA (P<0.05) than in controls. CONCLUSIONS: This study suggests that an imbalance in CTLA-4/CD28 expression or suppressed T-cell activity at the maternal-fetal interface may confer susceptibility to unexplained pregnancy loss.

[A/G polymorphism at position 49 in exon 1 of CTLA-4 gene in Chinese women with unexplained recurrent spontaneous abortion].

OBJECTIVE: To investigate whether A/G polymorphism at position 49 in exon 1 of cytotoxic T lymphocyte antigen-4 (CTLA-4) gene confers the susceptibility to unexplained recurrent spontaneous abortion in Chinese population. METHODS: One hundred and sixty-eight restrictive Chinese women with unexplained recurrent spontaneous abortion (URSA) and 117 women with normal pregnancy as control were included in this study. Polymerase chain reaction restrictive fragment length polymorphism (PCR-RFLP) was used to detect the polymorphism at position 49 in exon 1 of CTLA-4 gene. The frequency of alleles G/A, genotypes AA/AG/GG and phenotypes A+ (AA + AG)/G+ (GG + AG) of CTLA-4 were compared between URSA patients and controls. RESULTS: The different distributions of alleles G/A, genotype AA/AG/GG and phenotypes A+/G+ of CTLA-4 were observed between URSA patients and controls. The frequencies of both G allele [68.4% (230/336) vs 59.4% (139/234), P < 0.05] and GG genotype [48.8% (82/168) vs 33.3% (39/117), P < 0.05] were significantly higher in URSA group than that in control group, while the frequencies of AG genotype [39.3% (66/168) vs 52.1% (61/117), P < 0.05] and A+ (AA + AG) phenotype [51.2% (86/168) vs 66.7% (78/117), P < 0.05] were significantly lower in URSA group than that in control group. CONCLUSIONS: The results suggest that A/G polymorphism in exon-1 of CTLA-4 might confer the susceptibility to RSA in Chinese women.

A controlled spinal cord contusion for the rhesus macaque monkey.

Most in vivo spinal cord injury (SCI) experimental models use rodents. Due to the anatomical and functional differences between rodents and humans, reliable large animal models, such as non-human primates, of SCI are critically needed to facilitate translation of laboratory discoveries to clinical applications. Here we report the establishment of a controlled spinal contusion model that produces severity-dependent functional and histological deficits in non-human primates. Six adult male rhesus macaque monkeys underwent mild to moderate contusive SCI using 1.0 and 1.5mm tissue displacement injuries at T9 or sham laminectomy (n=2/group). Multiple assessments including motor-evoked potential (MEP), somatosensory-evoked potential (SSEP), MR imaging, and monkey hindlimb score (MHS) were performed. Monkeys were sacrificed at 6 months post-injury, and the lesion area was examined for cavitation, axons, myelin, and astrocytic responses. The MHS demonstrated that both the 1.0 and 1.5mm displacement injuries created discriminative neurological deficits which were severity-dependent. The MEP response rate was depressed after a 1.0mm injury and was abolished after a 1.5mm injury. The SSEP response rate was slightly decreased following both the 1.0 and 1.5mm SCI. MRI imaging demonstrated an increase in T2 signal at the lesion site at 3 and 6months, and diffusion tensor imaging (DTI) tractography showed interrupted fiber tracts at the lesion site at 4h and at 6 months post-SCI. Histologically, severity-dependent spinal cord atrophy, axonal degeneration, and myelin loss were found after both injury severities. Notably, strong astrocytic gliosis was not observed at the lesion penumbra in the monkey. In summary, we describe the development of a clinically-relevant contusive SCI model that produces severity-dependent anatomical and functional deficits in non-human primates. Such a model may advance the translation of novel SCI repair strategies to the clinic.

Location- and Subunit-Specific NMDA Receptors Determine the Developmental Sevoflurane Neurotoxicity Through ERK1/2 Signaling.

It is well established that developmental exposure of sevoflurane (an inhalational anesthetic) is capable of inducing neuronal apoptosis and subsequent learning and memory disorders. Synaptic NMDA receptors activity plays an essential role in cell survival, while the extra-synaptic NMDA receptors activation is usually associated with cell death. However, whether synaptic or extra-synaptic NMDA receptors mediate developmental sevoflurane neurotoxicity is largely unknown. Here, we show that developmental sevoflurane treatment decreased NR2A, but increased NR2B subunit expression both in vitro and in vivo. Sevoflurane-induced neuronal apoptosis was attenuated by synaptic NMDA receptors activation or low dose of exogenous NMDA in vitro. Interestingly, these effects could be abolished by NR2A inhibitor PEAQX, but not NR2B inhibitor Ifenprodil in vitro. In contrast, activation of extra-synaptic NMDA receptors alone had no effects on sevoflurane neurotoxicity. In the scenario of extra-synaptic NMDA receptors stimulation, however, sevoflurane-induced neuronal apoptosis could be prevented by addition of Ifenprodil, but not by PEAQX in vitro. In addition, sevoflurane neurotoxicity could also be rescued by memantine, an uncompetitive antagonist for preferential blockade of extra-synaptic NMDA receptors both in vitro and in vivo. Furthermore, we found that developmental sevoflurane-induced phospho-ERK1/2 inhibition was restored by synaptic NMDA receptor activation (in vitro), low dose of NMDA (in vitro) or memantine (in vivo). And the neuroprotective role of synaptic NMDA activity was able to be reversed by MEK1/2 inhibitor U0126 in vitro. Finally, administration of memantine or NMDA significantly improved spatial learning and memory dysfunctions induced by developmental sevoflurane exposure without influence on locomotor activity. These results indicated that activation of synaptic NR2A-containing NMDA receptors, or inhibition of extra-synaptic NR2B-containing NMDA receptors contributed to the relief of sevoflurane neurotoxicity, and the ERK1/2 MAPK signaling may be involved in this process.

Thermal nociception using a modified Hargreaves method in primates and humans.

Nociception is an important protective mechanism. The Hargreaves method, which involves measuring withdrawal latency following thermal stimulation to Thermal nociception using a modified Hargreaves method in primates and humans the paw, is commonly used to measure pain thresholds in rodents. We modified this technique to measure pain thresholds in monkeys and humans. The modified Hargreaves method was used to quantitate pain sensitivity in eight normal rhesus monkeys, 55 human volunteers, and 12 patients with spinal cord or cauda equina lesions. Thermal stimulation was delivered at 80% of maximum output, and the duration of the stimulation was set at a maximum of 10 seconds to avoid skin injury. The following withdrawal latencies were recorded: 2.7 ± 0.12 seconds in volunteers and 3.4 ± 0.35 seconds in neurologically intact monkeys (p>0.05). Patients with spinal cord or cauda equina lesions showed significantly increased latencies (p<0.001). The modified Hargreaves technique is a safe and reliable method that can provide a validated measure of physiological pain sensation.