Therapeutic effects of melatonin on heatstroke-induced multiple organ dysfunction syndrome in rats.

Melatonin reportedly exerts beneficial effects to attenuate multiple organ dysfunction syndrome (MODS) in septic shock. Heatstroke resembles septic shock in many aspects. Thus, this study was performed on the anesthetized rats by using heat exposure to induce heatstroke-associated MODS. We evaluated the effect of melatonin, a versatile molecule synthesized in the pineal gland and in many organs, in heatstroke rats and showed that melatonin (0.2-5.0 mg/kg of body weight, i.v., immediately after the start of heat stress) significantly (i) attenuated hyperthermia, hypotension and hypothalamic ischemia and hypoxia, (ii) reduced plasma index of the toxic oxidizing radicals like nitric oxide metabolites and hydroxyl radicals, (iii) diminished plasma index of hepatic and renal dysfunction like creatinine, blood urea nitrogen, alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, and lactate dehydrogenase, (iv) attenuated plasma systemic inflammation response molecules like soluble intercellular and lesion molecule-1, E-selectin, tumor necrosis factor-alpha, interleukin (IL)-1ß, and IL-6, (v) promoted plasma levels of an anti-inflammatory cytokine IL-10, (vi) reduced an index of infiltration of polymorphonuclear neutrophils in the lung like myeloperoxidase activity, and (vii) promoted the survival time to fourfold compared with the heatstroke alone group. Thus, melatonin could be a novel agent for the treatment of heatstroke animals or patients in the early stage.

Spinal Cord Lateral Hemisection and Asymmetric Behavioral Assessments in Adult Rats.

Incomplete spinal cord injury (SCI) often leads to impairments of sensorimotor functions and is clinically the most frequent type of SCI. Human Brown-Séquard syndrome is a common type of incomplete SCI caused by a lesion to one half of the spinal cord which results in paralysis and loss of proprioception on the same (or ipsilesional) side as the injury, and loss of pain and temperature sensation on the opposite (or contralesional) side. Adequate methodologies for producing a spinal cord lateral hemisection (HX) and assessing neurological impairments are essential to establish a reliable animal model of Brown-Séquard syndrome. Although lateral hemisection model plays a pivotal role in basic and translational research, standardized protocols for creating such a hemisection and assessing unilateralized function are lacking. The goal of this study is to describe step-by-step procedures to produce a rat spinal lateral HX at the 9th thoracic (T9) vertebral level. We, then, describe a combined behavior scale for HX (CBS-HX) that provides a simple and sensitive assessment of asymmetric neurological performance for unilateral SCI. The CBS-HX, ranging from 0 to 18, is composed of 4 individual assessments which include unilateral hindlimb stepping (UHS), coupling, contact placing, and grid walking. For CBS-HX, the ipsilateral and contralateral hindlimbs are assessed separately. We found that, after a T9 HX, the ipsilateral hindlimb showed impaired behavior function whereas the contralateral hindlimb showed substantial recovery. The CBS-HX effectively discriminated behavioral functions between ipsilateral and contralateral hindlimbs and detected temporal progression of recovery of the ipsilateral hindlimb. The CBS-HX components can be analyzed separately or in combination with other measures when needed. Although we only provided visual descriptions of the surgical procedures and behavioral assessments of a thoracic HX, the principle may be applied to other incomplete SCIs and at other levels of the injury.

Central projections and connections of lumbar primary afferent fibers in adult rats: effectively revealed using Texas red-dextran amine tracing.

Signals from lumbar primary afferent fibers are important for modulating locomotion of the hind-limbs. However, silver impregnation techniques, autoradiography, wheat germ agglutinin-horseradish peroxidase and cholera toxin B subunit-horseradish peroxidase cannot image the central projections and connections of the dorsal root in detail. Thus, we injected 3-kDa Texas red-dextran amine into the proximal trunks of L4 dorsal roots in adult rats. Confocal microscopy results revealed that numerous labeled arborizations and varicosities extended to the dorsal horn from T12-S4, to Clarke's column from T10-L2, and to the ventral horn from L1-5. The labeled varicosities at the L4 cord level were very dense, particularly in laminae I-III, and the density decreased gradually in more rostral and caudal segments. In addition, they were predominately distributed in laminae I-IV, moderately in laminae V-VII and sparsely in laminae VIII-X. Furthermore, direct contacts of lumbar afferent fibers with propriospinal neurons were widespread in gray matter. In conclusion, the projection and connection patterns of L4 afferents were illustrated in detail by Texas red-dextran amine-dorsal root tracing.

Inhibitory Effect of Endomorphin-2 Binding to the µ-Opioid Receptor in the Rat Pre-Bötzinger Complex on the Breathing Activity.

Opiates are commonly used analgesics that often cause clinical respiratory depression. However, their underlying mechanisms remain unclear. Endomorphin-2 (EM2) is a novel, endogenous tetrapeptide opioid with very high affinity and selectivity for the µ-opioid receptor (MOR). The pre-Bötzinger complex (pre-BötC) is considered the center of respiratory rhythm generation, and the synaptic connections in this region are essential for respiratory rhythm. The present study identified EM2-like immunoreactive (LI) axonal terminals in the pre-BötC of adult rats. Some EM2-LI axonal terminals made principally symmetric synapses with neurokinin 1 receptor (NK1R)-LI or MOR-LI neuronal dendritic processes in the pre-BötC. Unilateral microinjection of EM2 into the pre-BötC decreased breathing frequency and amplitude. A prior microinjection of the selective MOR antagonist ß-funaltrexamine (ß-FNA) into the pre-BötC prevented the effects of EM2. The present results suggest that EM2-LI axonal terminals modulate NK1R-expressing neurons in the pre-BötC and that EM2 plays a role in respiratory depression through MORs in the pre-BötC.

Crosstalk between Activated Microglia and Neurons in the Spinal Dorsal Horn Contributes to Stress-induced Hyperalgesia.

Stress has been shown to enhance pain sensitivity resulting in stress-induced hyperalgesia. However, the underlying mechanisms have yet to be elucidated. Using single-prolonged stress combined with Complete Freund's Adjuvant injection model, we explored the reciprocal regulatory relationship between neurons and microglia, which is critical for the maintenance of posttraumatic stress disorder (PTSD)-induced hyperalgesia. In our assay, significant mechanical allodynia was observed. Additionally, activated neurons in spinal dorsal horn were observed by analysis of Fos expression. And, microglia were also significantly activated with the presence of increased Iba-1 expression. Intrathecal administration of c-fos antisense oligodeoxynucleotides (ASO) or minocycline (a specific microglia inhibitor) attenuated mechanical allodynia. Moreover, intrathecal administration of c-fos ASO significantly suppressed the activation of neurons and microglia. Interestingly, inhibition of microglia activation by minocycline significantly suppressed the activation of both neurons and microglia in spinal dorsal horn. P38 inhibitor SB203580 suppressed IL-6 production, and inhibition of IL-6 receptor (IL-6R) activation by tocilizumab suppressed Fos expression. Together, our data suggest that the presence of a "crosstalk" between activated microglia and neurons in the spinal dorsal horn, which might contribute to the stress-induced hyperactivated state, leading to an increased pain sensitivity.

MicroRNA profiling identifies MiR-195 suppresses osteosarcoma cell metastasis by targeting CCND1.

Metastasis is a leading cause of mortality for osteosarcoma patients. The molecular pathological mechanism remains to be elucidated. In the previously study, we established two osteosarcoma cell lines with different metastatic potentials. Differential expressed genes and proteins regarding metastatic ability have been identified. MicroRNAs are important regulators in tumorigenesis and tumor progression. In this study, microRNA microarray was used to assess the differential expressed miRNAs level between these two cell lines. One of the top ranked miRNAs-miR-195 was identified highly expressing in lowly metastatic cells. It was showed that over-expression of miR-195 substantially inhibits migration and invasion of osteosarcoma cells in vitro and pulmonary metastasis formation in vivo. Meanwhile, CCND1 was identified as the target gene of miR-195 and further studied. More importantly, using real-time PCR, we evaluated the expression of miR-195 and CCND1 in osteosarcoma samples from 107 frozen biopsy tissues and 99 formalin- or paraformalin-fixed, paraffin-embedded (FFPE) tissues. Results indicated lowly expressed miR-195 or highly CCND1 correlated with positive overall survival and their expression inversely related to each other. In summary, our study suggests miR-195 functions as a tumor metastasis suppressor gene by down-regulating CCND1 and can be used as a potential target in the treatment of osteosarcoma.

Experimental treatment of radiation pneumonitis with human umbilical cord mesenchymal stem cells.

OBJECTIVE: To evaluate of the curative effect of human umbilical cord mesenchymal stem cells (hUC-MSCs) on rat acute radiation pneumonitis. METHODS: Fourty rats were randomly divided into control group, radiation group, stem cell prevention group, stem cell treatment group and prednisone treatment group. All rats except those in the control group were radiated with X ray to establish the acute radiation pneumonitis damage model. The hUC-MSCs cultured in vitro was administrated to the rats of the prevention group via tail vein (1×10(6) cells/kg BW) 24 h before the radiation, while the same administration was performed in the rats of the treatment group 24 h after the radiation. After 24 h post the radiation, the rats in the radiation group were given 0.4 mL physiological saline, and those in the prednisone group were given 1 mg/kg prednisone. All rats were observed and executed 72 h after the radiation to detect lung histological changes. RESULTS: After the administration of hUC-MSCs, the survival status of the rats in the prevention group and treatment group was obviously better than that in the control group. As shown by the histological staining, the morphology, proliferation activity and bronchial state of lung tissues were better in the prevention group and treatment group than in the control group. CONCLUSIONS: The hUC-MSCs have definite therapeutic effects on acute radiation pneumonitis in rats.

Expression change of TNF-α in myocardium and hepatic tissue of rats with compound stress of hyperthermia and lipopolysaccharide.

OBJECTIVE: To investigate the expression characteristics of TNF-α in myocardium and hepatic tissue of rats with compound stress of hyperthermia and lipopolysaccharide (LPS). METHODS: Male SPF Wistar rats were randomly divided into room temperature+physiological saline group (Group C), hyperthermia+physiological saline group (Group H), room temperature+LPS group (Group L) and hyperthermia+LPS group (Group HL). The rats were put in simulated climate cabin. Group HL and Group H were exposed in the environment at a dry bulb temperature (TDB) of (35.0±0.5) °C, while Group L and Group C were exposed in the environment at a TDB of (26.0±0.5) °C. The rats in Group HL and Group L were given tail intravenous injection of LPS 10 mg/kg, while the rats in Group H and Group C were given tail intravenous injection of 9 g/L NaCl 10 mL/kg. After the stress, immunohistochemical SABC staining method was used to detect the expression characteristics of TNF-α in myocardium and hepatic tissue of rats, and those rats were given routine pathological examinations. RESULTS: The expression of TNF-α in myocardium and hepatic tissue in Group HL was enhanced remarkably, and the tissue damages of Group HL were severest. CONCLUSIONS: The cardiotoxicity and hepatotoxicity caused by compound stress of hyperthermia and LPS is closely related to the expression of TNF-α.

An experimental study of preventing and treating acute radioactive enteritis with human umbilical cord mesenchymal stem cells.

OBJECTIVE: To test the curative effect of human umbilical cord-derived mesenchymal stem cells on rat acute radioactive enteritis and thus to provide clinical therapeutic basis for radiation sickness. METHODS: Human umbilical cord-derived mesenchymal stem cells were cultivated in vitro and the model of acute radioactive enteritis of rats was established. Then, the umbilical cord mesenchymal stem cells were injected into the rats via tail vein. Visual and histopathological changes of the experimental rats were observed. RESULTS: After the injection, the rats in the prevention group and treatment group had remarkably better survival status than those in the control group. The histological observations revealed that the former also had better intestinal mucosa structure, more regenerative cells and stronger proliferation activity than the latter. CONCLUSIONS: Human umbilical cord-derived mesenchymal stem cells have a definite therapeutic effect on acute radioactive enteritis in rats.

Human mesenchymal stem cells derived from limb bud can differentiate into all three embryonic germ layers lineages.

Mesenchymal stem cells (MSCs) have been isolated from many sources, including adults and fetuses. Previous studies have demonstrated that, compared with their adult counterpart, fetal MSCs with several remarkable advantages may be a better resource for clinical applications. In this study, we successfully isolated a rapidly proliferating cell population from limb bud of aborted fetus and termed them "human limb bud-derived mesenchymal stem cells" (hLB-MSCs). Characteristics of their morphology, phenotype, cell cycle, and differentiation properties were analyzed. These adherent cell populations have a typically spindle-shaped morphology. Flow cytometry analysis showed that hLB-MSCs are positive for CD13, CD29, CD90, CD105, and CD106, but negative for CD3, CD4, CD5, CD11b, CD14, CD15, CD34, CD45, CD45RA, and HLA-DR. The detection of cell cycle from different passages indicated that hLB-MSCs have a similar potential for propagation during long culture in vitro. The most novel finding here is that, in addition to their mesodermal differentiation (osteoblasts and adipocytes), hLB-MSCs can also differentiated into extramesenchymal lineages, such as neural (ectoderm) and hepatic (endoderm) progenies. These results indicate that hLB-MSCs have a high level of plasticity and can differentiate into cell lineages from all three embryonic layers in vitro.

Activated protein C can be used as a prophylactic as well as a therapeutic agent for heat stroke in rodents.

The present study was attempted to assess the prophylactic and the therapeutic effect of human recombinant activated protein C (APC; drotrecogin-alpha, activated) in experimental heat stroke. Anesthetized rats were divided into two groups and given vehicle solution 1 h before the start or immediately after the termination of heat stress (isotonic sodium chloride solution, 2 mL kg(-1) of body weight, i.v.) or APC (1-10 mg in 2 mL of isotonic sodium chloride solution per kilogram of body weight, i.v.). They were exposed to ambient temperature of 40 degrees C for 100 min to induce heat stroke. When the vehicle-pretreated rats underwent heat stress, their survival time values were found to be 57 to 71 min. Pretreatment or treatment with APC significantly increased survival time (122-221 min). All vehicle-pretreated heat stroke animals displayed systemic inflammation (evidenced by increased TNF-alpha, IL-1alpha, and IL-6) and activated coagulation (evidenced by increased levels of activated partial thromboplastin time, prothrombin time, and D-dimer and decreased levels of both platelet count and protein C). Biochemical assay also revealed that both renal and hepatic dysfunction (e.g., increased plasma levels of blood urea nitrogen, creatinine, adenine aminotransferase, aspartate aminotransferase, and alkaline phosphatase) were noted during heat stroke. A significant decrease in both cerebral blood flow and partial pressure of oxygen in hypothalamus were also observed in vehicle-pretreated heat stroke animals. These heat stroke reactions were all significantly reduced by pretreatment or treatment with human recombinant APC. The results indicate that human recombinant APC can be used as a prophylactic and a therapeutic agent for experimental heat stroke by ameliorating systemic inflammation, hypercoagulable state, and multiple organ dysfunction.

[Changes of serum urea and creatinine concentrations in rats with lipopolysaccharide and heat co-exposure].

OBJECTIVE: To investigate the effects of lipopolysaccharide (LPS) and heat co-exposure on serum urea and creatinine (Cr) concentrations in rats. METHODS: Male Wistar rats were randomized into normothermic saline injection control group (group C), heat exposure saline injection group (group H), normothermic LPS injection group (group L), and heat exposure LPS injection group (group HL). The rats in groups H and HL were exposed to heat in a chamber at an dry bulb temperature (Tdb) of 35.0-/+0.5 degrees Celsius, and those in groups C and L were kept in a chamber at Tdb of 26-/+0.5 degrees Celsius. LPS (8 mg/kg) was injected via the tail vein in the rats in groups L and HL to induce endotoxemia, while those in groups C and H were given normal saline injection (8 ml/kg) via the tail vein. The serum levels of urea and Cr were determined at the time points of 0, 40, 80, and 120 min after the injections. RESULTS: No significant difference was found in serum Cr level at any level of the main effects of time, drug, or Tdb (P>0.05), but serum urea level varied significantly between the different time points, different levels of Tdb, and the drug injections (P<0.01). Significant interactions were identified between the time after injection, injected agents, and Tdb (P<0.01). Except for those in the group C, all rats showed elevated serum urea levels 40 min after the injection, particularly those in group HL. The serum urea levels were positively correlated to the level of tumor necrosis factor-alpha (P<0.01). CONCLUSION: Co-exposure to LPS and heat in rats may elicit and worsen systemic inflammatory response syndrome and kidney injury.