Mechanism of myeloid differentiation factor 2 on mediating sepsis-associated encephalopathy / 中华危重病急救医学

OBJECTIVE@#To investigate the role and underlying mechanism of human myeloid differentiation protein 2 (MD2) in the process of neuronal death induced by lipopolysaccharide (LPS) by establishing an in vitro model of sepsis-associated encephalopathy (SAE) by LPS.@*METHODS@#Healthy C57BL/6J mice at 14-18 days of gestation were selected, and brain cortical tissue was taken from fetal mice. Neurons were stimulated with 0 (control), 1, 5 and 10 g/L of LPS for 24 hours. The release of lactate dehydrogenase (LDH) was detected and the death of neurons was observed. Enzyme-linked immunosorbent assay (ELISA) was used to detect the levels of inflammatory factors interleukins (IL-6, IL-1β), in order to determine the optimal dose of LPS for establishing an in vitro neuroinflammation model of SAE. The cells were divided into blank control group and LPS group. Terminal deoxynucleotidyl transferase-mediated dUTP nick-end-labeling (TUNEL) was used to discover apoptosis. Western blotting was used to detect the expression of the relevant protein markers activated caspase-3, necroptosis-associated protein neuronal receptor-interacting serine/threonine-protein kinase 3 (RIPK3) and phosphorylated RIPK3 (p-RIPK3). Immunofluorescence chemical staining was used to detect the expressions of p-RIPK3 and microtubule-associated protein 2 (MAP2) to evaluate the type of cell death and the degree of neuronal death. Western blotting was used to detect MD2 expression. Immunofluorescence chemical staining was performed to observe the expression and distribution of p-RIPK3 and MD2 in neurons to assess whether MD2 was involved in the inflammatory response promoting neuronal death. In addition, the cells were divided into blank control group, LPS group, and MD2 interfering peptide group (LPS+TC group), and the levels of IL-6, IL-1β and LDH were detected to evaluate whether interfering with MD2 can alleviate LPS induced neuroinflammation.@*RESULTS@#10 g/L LPS induced notable neuronal death, and the release of LDH in neurons stimulated with this concentration for 24 hours was significantly higher than that in the blank control group (relative release: 1.45±0.04 vs. 1.00±0.00, P < 0.01), indicating apoptosis and necroptosis occurred in neurons, and the levels of inflammatory factors IL-6 and IL-1β were remarkable increased [IL-6 (relative level): 1.94±0.04 vs. 1.00±0.00, IL-1β (relative level): 1.53±0.09 vs. 1.00±0.00, both P < 0.01]. Compared with the blank control group, the apoptosis of cells, cleaved-caspase-3 expression, the p-RIPK3/RIPK3 ratio, and p-RIPK3 expression around neurons in the LPS group were significantly increased [cleaved-caspase-3/GAPDH: 1.55±0.10 vs. 1.00±0.00, P < 0.01; p-RIPK3/RIPK3 ratio (relative value): 1.54±0.06 vs. 1.00±0.00, P < 0.05], which suggested that typical apoptosis and necroptosis apoptosis occurred in neurons in the septic environment. Furthermore, MD2 expression was significantly increased in the LPS group compared with the blank control group (MD2/GAPDH: 1.91±0.07 vs. 1.00±0.00, P < 0.01), and MD2 expression around neurons was increased, indicating that LPS-induced MD2 upregulation may play a key role in neuroinflammation and induction of neuronal death in sepsis. In addition, compared with the LPS group, the MD2-interfering peptide could reduce the expression levels of inflammatory factors IL-6 and IL-1β [IL-6 (relative level): 1.16±0.08 vs. 1.94±0.04, IL-1β (relative level): 1.15±0.05 vs. 1.75±0.09, both P < 0.01] and decrease LDH release (relative release: 1.09±0.01 vs. 1.44±0.04, P < 0.05).@*CONCLUSIONS@#LPS induced neuronal inflammatory responses via MD2, which ultimately leads to apoptosis and necroptosis. Interfering with MD2 reduces inflammation and inhibits neuronal death.

Recent progress of antimicrobial peptides in sepsis treatment / 中华危重病急救医学

Sepsis is a life-threatening organ dysfunction due to the dysregulation of host responses during infection. Severe systemic inflammatory response syndrome (SIRS) is the primary pathophysiological feature. Despite the classical antibiotic therapies play an important role in sepsis, the emergence of multi-resistant bacteria makes a greater challenge in clinical. Antimicrobial peptides (AMP) which consist of small cationic peptides, can be found in most organisms. As a result of their board-spectrum antibacterial activities and immunoregulatory functions, AMPs may have an excellent effect on the treatment of sepsis. In this review, we will discuss the basic role of AMPs in sepsis treatment and their application prospect and the challenges which need to be resolved in order to provide ideas for clinical application of AMPs.

How to improve the teaching ability of young teachers of critical care medicine? / 中华危重病急救医学

Intensive care unit (ICU) in teaching hospital plays important roles in teaching work. The young teachers of critical care medicine are gradually becoming the backbone of teaching work. Improving the teaching ability of young teachers is essential to increase learning motivation of the students and to promote the overall teaching quality of critical care medicine. Therefore, pay attention to help the young teachers of critical care medicine to improve their teaching skill is good for enhancing the faculty developing of critical care medicine, as well as essential for the prosperity and sustainable development of critical care medicine. Based on the problems existing during the teaching process of young teachers of critical care medicine and aimed to train excellent teachers, this article discussed the teaching methods and experience of young teachers of critical care medicine which focuses on teaching program design, class affinity improvement and humanistic education in order to improve the teaching level of young teachers of critical care medicine.

α7 nicotinic acetylcholine receptor agonist attenuated the lipopolysaccharide-induced inflammatory response via inhibiting the activation of nuclear factor-κB / 中华危重病急救医学

Objective To investigate the effects of α7 nicotinic acetylcholine receptor (α7nAChR) on the inflammatory response induced by lipopolysaccharide (LPS) in RAW264.7 macrophages and its molecular mechanisms. Methods RAW264.7 macrophages were culturedin vitro. Inflammatory cell model was constructed by LPS stimulation. Cells were challenged by LPS (1, 10, 100 and 500μg/L) for 5 hours or 100μg/L LPS for 0, 2, 4, 8, 12, 24, 48 and 72 hours, and the release of tumor necrosis factor-α (TNF-α) was detected by the enzyme linked immunosorbent assay (ELISA). The location of α7nAChR was examined in RAW264.7 macrophages by immunofluorescence. Then the cell proliferation and toxicity kit (CCK-8) was used to detect 1, 10, 100, 1000μmol/L GTS-21, a α7nAchR agonist, on the cell viability after LPS stimulation. ELISA was used to detect 1, 10, 100, 1000μmol/L GTS-21 on the levels of TNF-α, interleukin 1β (IL-1β) after LPS stimulation. Cells were challenged with 100μg/L LPS and 100μmol/L GTS-21, then, the level of high mobility group box 1 (HMGB1) was detected by Western Blot and the intracellular location of HMGB1 and nuclear factor-κB p65 (NF-κB p65) was tested by immunofluorescence.Results LPS increased the level of TNF-α to a peak at the concentration of 100μg/L and at 24 hours after stimulation. Theα7nAChR expressed on the macrophages. The cell viability was decreased in a dose-dependent manner [(96.2±1.0)%, (92.0±1.1)% vs. (86.5±2.2)%, bothP < 0.05]. Compared with the control group, the levels of TNF-α and IL-1βin the supernatant of LPS group were significantly increased [TNF-α (ng/L): 453.0±60.6 vs. 100.8±3.2, IL-1β(μg/L): 8.21±0.31 vs. 0.87±0.16, bothP < 0.05]. TNF-α and IL-1β were significantly decreased by 10μmol/L and 100μmol/L GTS-21 in a dose-dependent manner [TNF-α (ng/L): 227.5±17.5, 81.0±8.8 vs. 453.0±60.6;IL-1β (μg/L): 4.86±0.72, 2.32±0.45 vs. 8.21±0.31, allP < 0.05]. GTS-21 significantly reduced the expression of HMGB1 which was induced by LPS management (gray value: 0.788±0.130 vs. 2.061±0.330,P < 0.05) and reversed LPS-induced HMGB1 cytoplasmic transfer. GTS-21 also reversed LPS-induced nuclear translocation of NF-κB p65. Conclusion GTS-21 reduces the inflammatory response via inhibiting the activation of NF-κB.

Statistics of causes of death and analysis of risk factors in a surgical intensive care unit / 中华危重病急救医学

Objective To summarize the causes of death and to analyze the risk factors in a surgical intensive care unit (SICU).Methods The relevant information of patients died in the SICU of Xijing Hospital of Fourth Military Medical University in past 15 years (from December 1999 to February 2015) was retrospectively analyzed.The gender,age, reason and date of hospitalization, date of transfer SICU, past medical history, whether or not admitted directly from emergency department, or transferred from other department, operated or not, date of death, the main cause of death, acute physiology and chronic health evaluation Ⅱ (APACHE Ⅱ) score, the history of undergoing mechanical ventilation, continuous renal replacement therapy (CRRT), or antifungal therapy, as well as the ratio of the patients with body temperature higher than 39 ℃, white blood cell (WBC) count higher than 10 × 109/L, platelet (PLT) count below 100 × 109/L, albumin (Alb) below 35 g/L of two periods, namely from December 1999 to July 2007 (the first period),and from August 2007 to February 2015 (the second period) were compared.The above parameters were compared with those of 201 survivors in SICU, and the risk factors leading to death were analyzed by logistic regression.Results From December 1999 to February 2015, 4 317 patients were taken care of in the SICU.Among them, the number of death was 186, and the mortality rate was 4.3％.In the first time period (from December 1999 to July 2007), the total number of patients was 1 356, and the number of death were 109 (the mortality rate was 8.0％).In the second period, i.e.from August 2007 to February 2015, the number of SICU patients was 2 961, and 77 died (the mortality rate was 2.6％).The difference of mortality rate between the two periods was statistically significant (x2 =66.707, P =0.001).The death rate of patients transferred directly from emergency department in tle first period was 79.8％ (87/109), and it was lower in the second period (51.9％, 40/77, x2 =16.181, P =0.001).The death rate of the patients with blood Alb below 35 g/L in the second period (59.7％, 46/77) was higher than that of the first period (41.3％, 45/109, x2 =6.151, P =0.017).The top three causes of death from December 1999 to February 2015 were sepsis (38.2％), trauma (16.7％), and operation for cancer (14.0％).In the first period, the top three causes of death were sepsis (35.8％), trauma (22.0％),and operation for cancer (13.8％).In the second period, the top three causes of death were sepsis (41.6％), damage of the central nervous system (16.9％), and operation for cancer (14.3％).Top three reasons for SICU admission were trauma (29.03％), abdominal pain (20.97％) and other reasons (18.82％).Top three departments from which the patients were transferred were the emergency department (19.35％), orthopedics department (17.20％), and hepatobiliary department (16.13％).Logistic regression analysis showed that age [odds ratio (OR) =2.025, 95％ confidence interval (95％CI) =1.500-2.734, P =0.000], mechanical ventilation (OR =3.514, 95％CI =1.701-7.259, P =0.001), CRRT (OR =5.604,95％CI =3.003-10.459, P =0.000), body temperature higher than 39 ℃ (OR =1.992, 95％CI =1.052-3.771, P =0.034) were the risk factors of death in SICU patients.Conclusion Sepsis and severe trauma are the leading causes of death in severe SICU patients, to whom with risk factors of death enough attention should be given.

EXPRESSIONS OF IL-1? AND IL-1RI mRNA IN THE RAT CAROTID BODY / 解剖学报

Objective To study the expressions of IL-1 receptor type Ⅰ(IL-1RI) mRNA and IL-1? protein in the rat carotid body. Methods In situ hybridization,immunofluorescence double staining and Western blotting methods were used. Results The result of in situ hybridization showed that the positive signal of IL-1? mRNA was mainly located in the glomus cells of the carotid body.The result of immunofluorescence double staining showed that IL-1? protein also expressed in the glomus cells of the organ.The Western blotting proved that the IL-1? immunoreactive band appeared at 18kD,consistent with the molecular weight of the cytokine.Conclusion The glomus cells of the rat carotid body not only express IL-1RI mRNA,but also IL-1?.

RELATIONSHIP BETWEEN CCK-AND VP-NEURONS IN THE RETROCHIASMATIC AREA—DOUBLE LABELED IMMUNOELECTRON MICROSCOPIC STUDY / 解剖学报

The relationship between CCK-and VP-neurons in the rat retrochiasmatic area was studied at ultrastructural level by means of pre-embedding(PAP) double immunoelectron microscopic labeling technique. First, the VP-immunoreactivity was demonstrated by DAB method. After thoroughly washing, the CCK-immuno- reactivity was revealed by ammonium molybdate-TMB method. Being stabili- zed by DAB-cobalt chloride, the sections were embedded in Epon 812. Under ele- ctron microscope, it was observed that in the retrochiasmatic area, the VP-LI products distributed diffusely as high electron dense granular or flocculent depo- sits, whereas the CCK-LI products distributed sparsely as needle-or mass-like deposits. VP-LI perikarya were small in size with oval shape and CCK-LI peri- karya were medium in size with polygonal shape. CCK-LI perikarya and dendri- tes received afferent synapses from non-CCK- and non-VP-axonal terminals VP- LI axons received afferent synapses from VP and non-VP-axonal terminals It was interesting that the VP-LI axonal terminals formed efferent axoaxonic syna- pses with CCK-LI axonal endings and, vice versa, the CCK-LI axonal terminals established also efferent axoaxonic synapses with VP-LI axonal endings. The above mentioned results identified for the first time that in the rat retrochiasma- tic area not only there were CCK- and VP-neurons, but also there were reciprocal synaptic regulations between above two kinds of peptidergic neuron, providing new ultrastructural basis for the regulatory mechanism of the neuroendocrine in hypothalamus.

THE AUTOREGULATORY SYNAPSES OF SP-NEURONS IN THE NUCLEUS RAPHE MAGNUS OF RATS / 解剖学报

Using immunoelectron microscopic technique the SPergic autoregulatory synapses in the nucleus raphe magnus were studied. The results showed that there were SPlike perikarya and nerve fibers. The positive perikarya mainly were large polypolar cells. The positive fibers appeared as beaded-like and formed axodendritic synapses with SP-positive dendrites besides synapses formed with SP-negative structures. In the autoregulatory synapses the pre- and postsynaptic element were both labeled with immunoreactive products which precipitated at the periphery of small clear synaptic vesicles and the dendritic tubes, on the outer membrane of mitochondria, and in the cytoplasmic matrix. The pre- and postsynaptic membrane were symmetrical, and the synaptic vesicles aggregated near the presynaptic membrane. The synaptic cleft was about 20 nm in width and contained electron dense materials. The generality, structural characteristics and functional significance of the autoregulatory sysnapses were discussed.

THE RELATIONSHIP BETWEEN NT- AND NPY-NEURONS IN THE ARCUATE NUCLEUS A DOUBLE LABELING IMMUNOELECTRON MICROSCOPIC STUDY / 解剖学报

The distribution of neurotensin (NT) and neuropeptide Y (NPY) in the rat hypothalamic arcuate nucleus has been studied ultrastructurally by means of double labeling preembedding immunoelectron microscopic PAP technique. First, the NPY immunoreaction was demonstrated by chromogen DAB, and second, the NT immunoreaction was demonstrated by ammonium molybdate-TMB method. After being stabilized by DAB-cobalt chloride, the vibratome sections were processed for electron microscopic study. The results showed that in the arcuate nucleus the NPY immunoreactive products appeared as high electron-dense granular or flocculent materials deposited diffusely in the organelles and matrix of perikaryon, around the dendritic microtubules and axonic small clear vesicles. Whereas the NT immunoreactive products were dense needle- or mass-like deposits distributed dispersively in the perikaryon, dendrites and axon terminals. They can easily be distinguished although being intermingled together. The NPY-containing dendrites and axons formed synaptic connections with immuno-negative axon terminals, NT-containing somata and dendrites formed also synaptic conections with negative axon terminals. In addition, NPY-positive axon terminals formed symmetrical axodendritic synapses with NT-positive dendrites. The present results provided another new ultrastructural evidence for the peptidergic synaptic regulation of NT neurons in hypothalamus.

A DOUBLE LABELED IMMUNOELECTRON MICROSCOPIC STUDY OF NEUROTENSINERGIC AND SUBSTANCE P-ERGIC STRUCTURES IN THE RAT ARCUATE NUCLEUS / 解剖学报

The distributions of neurotensin(NT) and substance P(SP) in the arcuate nucleus of rat hypothalamus have been studied by means of double labeled pre-embedding immunoelectron microscopic technique. It was observed that there were SP- and NT-containing dendrites, perikarya and axons in the arcuate nucleus. SP- and NT-containing dendrites and axons received asymmetric afferent synapses from immunonegative axons. SP-positive axonal terminals established symmetric axo-somatic and axo-dendritic synapses with immunonegative perikarya and dendrites as well as symmetric axo-somatic synapses with NT-positive perikarya. The results of this study directly indicate for the first time that the NT-ergic neurons in rat arcuate nucleus receive innervation from SP-ergic neurons, and provided an ultrastructural evidence for the synaptic regulation of the neuroendocrine of the hypothalamus.

ELECTRON MICROSCOPIC STUDY OF THE RELATIONSHIP BETWEEN CATECHOLAMINERGIC AND PEPTIDERGIC NERVE TERMINALS IN THE RAT NEUROHYPOPHYSIS / 解剖学报

In order to clarify the regulatory mechanism of the neurohormone releasing in the neurohypophysis, the immunohistochemical and chemical lesion method were combined to demonstrate the vasopressin (VP)-and catecholamine (CA)-containing nerve terminals, and their distribution and relationship were observed under electron microscopic level. The results showed that in the rat neurohypophysis there were not only widely distributed VP nerve terminals, but also there were many 6-OHDA induced degenerated nerve endings. The close relationship even synapse-like contacts existed between the CA-ergic endings and pituicytes as well as microglial cells. It was very interesting that the CA-ergic boutons formed axoaxonic synapses with VP-containing boutons. In this case, the CA-bouton was presynaptic element whereas the VP-bouton served as postsynaptic element. The above mentioned results probably provided ultrastructural evidence for the regulatory mechanism of the neurohormone releasing in the neurohypophysis for the first time.

AN ELECTRON IMMUNOCYTOCHEMICAL STUDY OF CHOLECYSTOKININ-CONTAINING NEURONS IN THE HYPOTHALAMIC PARAVENTRICULAR NUCLEUS / 解剖学报

Using electron immunocytochemical method, the ultrastructural distribution and the synaptic connections of CCK-containing neurons in the paraventricular nucleus (PVN) of the rat were studied. The results showed that the CCK-like immunoreactive products located in farge granular vesicles, cytoplasmic matrix, at the periphery of small clear vesicles, rough endoplasmic reticulum and the membrane of mitochondria. The CCK-positive nerve cell bodies were large or small in size and distributed mainly in the medial part of the PVN, subependymal region and the vicinity of capillaries. Some of them as postsynaptic elements formed axosomatic synapses with CCK-negative axonal terminals. The CCK-positive dendrites and axons situated everywhere in the PVN. Some of them as postsynaptic elements formed axodendritic and axoaxonic synapses with CCK-negative structures. Some CCK-positive axonal endings surrounded the capillaries. Other CCK axonal terminals as presynaptic elements formed axosomatic, axondendritic and axo-axonic synapses with CCK-negative structures, respectively. In addition, we have first found that the CCK-positive dendrites penetrated ependyma and contacted directly with the cerebrospinal fluid in third ventricle, the CCK-positive axons traveled in the cavity of third ventricle near the ependyma. The above mentioned results suggested: (1) the soma, dendrite and axon of the CCK-containing neurons and CCK-negetive neurons in the PVN might form local neuronal circuit; (2) the neuron vessel circuit might be established between CCK-containing neurons and the blood vessels in the PVN; (3) the CSFcontacting neurons in the PVN may participate in forming brain-cerebrospinal fluid neurohumoral circuit and regulate functional activity of distal target area through the CSF pathway.

DESCENDING PROJECTIONS OF SUBSTANCE P-AND CHOLECYSTOKININ-CONTAINING NEURONS OF THE PERIAQUEDUCTAL GRAY AND EDINGER-WESTPHAL NUCLEUS TO THE SPINAL CORD——A STUDY WITH THE COMBINING METHOD OF TRACING WITH IMMUNOCYTOCHEMISTRY / 解剖学报

Projections from substance P (SP)-and cholecystokinin (CCK) containing neurons in the periaqueductal gray(PAG)and Edinger-Westphal(E-W)nucleus to the spinal cord were studied by means of the combining method of HRP tracing with immunocytochemistry in rats. The results showed that a few neurons in the ventrolateral region of PAG projected bilaterally to the cervical, thoracic and lumbar segments of the spinal cord, with the predominant projections from the ipsilateral side. The authors reported that these descending projection neurons showed SP-like immunoreactivity for the first time(account for 48%). The neurons of E-W nucleus projected diffusely to all segments of the spinal cord contained SP (70%) or CCK (73%) respectively, suggesting that at least a part of E-W neurons projecting to the spinal cord contain both SP and CCK.