大鼠坐骨神经结扎后脊髓钙结合蛋白PV的表达变化

目的:研究大鼠坐骨神经结扎模型钙结合蛋白Parvalbumin(PV)在脊髓的时空变化规律,为探讨其在神经再生中的作用与机制提供实验依据。方法:SD大鼠随机分为假手术对照组和坐骨神经结扎组,实验组结扎后分别存活1,3,7,14或21d,采用免疫组化结合图像分析技术观察PV在脊髓的表达变化。结果:在对照组,PV免疫阳性神经元主要分布于腰髓背角Ⅱ层,Ⅲ～Ⅵ层只观察到少量散在分布的PV样阳性神经元,脊髓前角Ⅷ层和Ⅸ层内也可见少量多极的大型阳性神经元。术后各时间点PV样阳性神经元表达下降,14d下降最显著,21d表达有所上升,但还是低于7d组。脊髓后角PV免疫阳性产物灰度值测定结果显示:术后14d后角PV表达最低,与对侧和对照组以及1、3d组相比有统计学意义(P<0.05)。结论:坐骨神经结扎后PV表达变化呈现一定的时空模式,为进一步揭示PV在神经系统疾病中的作用提供实验依据。     

脊髓外科手术术后精神障碍患者发病影响因素及抑制性神经递质水平、神经营养因子表达变化情况分析

摘要 目的：探讨与分析脊髓外科手术术后精神障碍患者发病影响因素及抑制性神经递质水平、神经营养因子表达变化情况。方法：选择2016年9月到2021年5月本院完成脊髓外科手术的患者83例作为研究对象,检测血清抑制性神经递质水平、神经营养因子（NTFs）表达水平。所有患者都给予抑郁自评量表（SDS）调查、执行功能行为评定量表成人版自评问卷（BRIEF-A）评分并进行相关性分析。结果：83例患者术后平均SDS评分为45.10±2.87分,判定为精神障碍23例（精神障碍组）,占比27.7 %。精神障碍组的性别、年龄、手术时间、术中出血量与非精神障碍组对比无差异（P>0.05）,精神障碍组的饮酒、术后清醒时间与非精神障碍组对比有差异（P<0.05）。精神障碍组的BRI自我控制、情感控制、转移、抑制等评分与MI任务启动、任务监督、工作记忆、计划、组织评分都高于非精神障碍组（P<0.05）。精神障碍组的血清NTFs含量低于非精神障碍组,血清HA与5-HT含量高于非精神障碍组（P<0.05）。在83例患者中,Pearson分析显示SDS评分与饮酒、术后清醒时间、血清NTFs、NA、5-HT含量都存在相关性（P<0.05）;二分类logistic逐步回归显示术后清醒时间、血清NTFs、NA、5-HT含量都为导致脊髓外科手术术后精神障碍患者发病的重要因素（P<0.05）。结论：脊髓外科手术术后精神障碍的发生较常见,可导致患者认知与执行功能降低,多伴随有抑制性神经递质水平表达上升与神经营养因子表达下降,血清NTFs、NE、5-HT含量都为导致精神障碍发病的重要因素。     

Substance P antagonist-induced spinal cord vasoconstriction: Effects of thyrotropin-releasing hormone and substance P agonists

Local spinal cord vasomotor effects of 3 substance P (SP) antagonists were studied in the rat following intrathecal (IT) administration. Each SP antagonist (3.3 nmol) increased spinal cord vascular resistance and reduced blood flow. A LH-RH antagonist analog (10 nmol) of similar molecular weight and which also contained multiple D-Trp residues did not cause spinal cord vasoconstriction. The vasoconstrictor action of the SP antagonist, [D-Arg¹, D-Pro², D-Trp^7,9, Leu¹¹]-SP ([D-Arg]-SP) was unaffected by pretreatment with a stable SP receptor agonist (5 nmol IT). Given evidence for a cerebral vasodilator action of TRH agonists, the effects of TRH (IV) and a stable TRH analog (MK-771, IT) on [D-Arg]-SP-induced vasoconstriction were also assessed. Neither TRH nor MK-771 prevented the [D-Arg]-SP-induced vasoconstriction. However, TRH (IV) but not MK-771 (IT) partially opposed [D-Arg]-SP-induced reduction in thoracic spinal cord blood flow. Thus, SP antagonists cause spinal cord vasoconstriction by a non-SP receptor mediated phenomenon. In addition, the attenuation of SP-antagonist-induced neuropathological changes previously reported with IV. TRH administration is likely due to less severe consequences of vasoconstriction in the presence of a higher initial baseline blood flow rather than direct prevention of the vasoconstriction.     

Hindlimb paralytic effects of arginine vasopressin and related peptides following spinal subarachnoid injection in the rat

Intrathecal (IT) injection of arginine vasopressin (AVP) in rats caused a transient (<30 min), dose-related paralysis of the hindlimbs, loss of hindlimb and tail nociceptive responsiveness, and increased mean arterial pressure. Motor dysfunction was produced with comparable potency by lysine vasopressin (LVP) and arginine vasotocin (AVT); oxytocin (OXY) was approximately 1000 times less potent. Paralysis induced by these peptides was selectively blocked following IT pretreatment with 0.5 nmoles of the vasopressin V₁ receptor antagonist [1-(β-mercapto-β,β-cyclopentamethylene propioinic acid), 2-(O-methyl)tyrosine] Arg⁸-vasopressin (d(CH₂)₅[Tyr(Me²)]AVP). Pressor and antinociceptive responses to AVP were also blocked by this compound. However, at higher doses (2–5 nmoles, IT), d(CH₂)₅[Tyr(Me²)]AVP produced hindlimb paralysis, antinociception, and pressor responses by itself. In contrast to the fiber degeneration, cell loss, and necrosis found in lumbosacral cords of rats persistently paralyzed by other peptides (dynorphin A, somatostatin, and ICI 174864), neuropathological changes were not evident in spinal cords of rats transiently paralyzed by IT AVP. These results indicate that AVP-related peptides affected diverse spinal cord functions through interactions with a V₁-like receptor. The similar pattern of cardiovascular and antinociceptive responses to other peptides (dynorphin A, somatostatin, and ICI 174864), which also caused hindlimb paralysis, suggests that the former responses may actually reflect the nonselective consequences of a peptide-induced disruption of spinal cord function, rather than specific shared pharmacological effects.     

大鼠颈段脊髓灰质板层及其细胞构筑

取12只SD(Sprague-Dawley)大鼠颈段脊髓横断面冰冻切片,用焦油紫和Pal-Weigert染色法染色,光镜观察SD大鼠颈段脊髓的组织结构,对SD大鼠脊髓灰质板层的细胞构筑进行研究。结果显示,Ⅰ层边界呈长弧形,Ⅰ、Ⅱ、Ⅲ、Ⅳ层呈层叠状排列,细胞多为椭圆形。Ⅴ、Ⅵ层均可分为内侧部和外侧部,外侧部分布大型细胞,Ⅴ层内侧部以中型椭圆形、三角形细胞为主,Ⅵ层内侧部以中型梭形细胞为主。Ⅶ层位于中间带,C5-C7Ⅶ层向前角延伸和Ⅷ层一起占据前角大部,Ⅶ及Ⅷ层的中型和大型细胞呈集中分布。Ⅷ层在C1-C4及C8占前角大部,在C5-C7位于前角内侧部。Ⅸ层主要由含大型运动神经元的核团组成。Ⅹ层由中型梭形细胞和小型星状细胞组成。对比观察发现,SD大鼠脊髓颈段板层类似于猫的Rexed分层,但灰质轮廓、板层出现节段、板层形态及其变化等方面均有所不同。     

Detection of specific antibodies in cord blood, infant and maternal saliva and breast milk to staphylococcal toxins implicated in sudden infant death syndrome (SIDS)

The common bacterial toxins hypothesis of sudden infant death syndrome (SIDS) is that nasopharyngeal bacterial toxins can trigger events leading to death in infants with absent/low levels of antibody that can neutralise the toxins. The aim of this study was to investigate nasopharyngeal carriage of Staphylococcus aureus and determine levels of immunity in the first year of life to toxic shock syndrome toxin (TSST-1) and staphylococcal enterotoxin C (SEC). Both toxins have been implicated in SIDS cases. Seventy-three mothers and their infants (39 males and 34 females) were enrolled onto the study. The infants had birth dates spread evenly throughout the year. In infants, S. aureus carriage decreased significantly with age (P<0.001). Between 40% and 50% of infants were colonised with S. aureus in the first three months of life and 49% of the isolates produced one or both of the staphylococcal toxins. There was a significant correlation between nasopharyngeal carriage of S. aureus in mothers and infants in the three months following the birth (P<0.001). Carriage of S. aureus in infants and their mothers was not significantly associated with levels of antibody to TSST-1 or SEC in cord blood, adult saliva or breast milk. Infants colonised by S. aureus had higher levels of salivary IgA to TSST-1 than infants who were culture negative. Analysis of cord blood samples by a quantitative ELISA detected IgG bound to TSST-1 and SEC in 95.5% and 91.8% of cases respectively. There was a marked variation in levels of maternal IgG to both TSST-1 and SEC among cord blood samples. Maternal age, birth weight, and seasonality significantly affected the levels of IgG binding to TSST-1 or SEC. Analysis of infant saliva samples detected IgA to TSST-1 and SEC in the first month after birth; 11% of samples tested positive for salivary IgA to TSST-1 and 5% for salivary IgA to SEC. By the age of two months these proportions had increased to 36% and 33% respectively. More infants who used a dummy tested positive for salivary IgA to TSST-1 compared to infants who did not use a dummy. Levels of IgA to TSST-1 and SEC detected in the breast-milk samples varied greatly among mothers. There was a trend for infants receiving breast milk with low levels of antibody to TSST-1 or SEC to have higher levels of salivary antibody to the toxins. In conclusion, passive immunity to toxins implicated in SIDS cases varies greatly among infants. Infants are able to mount an active mucosal immune response to TSST-1 and SEC in the first month of life.     

Synaptic interactions between nonspiking local interneurones in the terminal abdominal ganglion of the crayfish

Nonspiking local interneurones are the important premotor elements in arthropod motor control systems. We have analyzed the synaptic interactions between nonspiking interneurones in the crayfish terminal (6th) abdominal ganglion using simultaneous intracellular recordings. Only 15% of nonspiking interneurones formed bi-directional excitatory connections. In 77% of connections, however, the nonspiking interneurones showed a one-way inhibitory interaction. In these cases, the presynaptic nonspiking interneurones received excitatory synaptic inputs from the sensory afferents innervating hairs on the surface of the uropods and the postsynaptic nonspiking interneurones received inhibitory synaptic inputs that were partly mediated by the inputs to the presynaptic nonspiking interneurones. The membrane hyperpolarization of the postsynaptic nonspiking interneurones mediated by the presynaptic nonspiking interneurones was reduced in amplitude when the hyperpolarizing current was injected into the postsynaptic interneurones, or when the external bathing solution was replaced with one containing low calcium and high magnesium concentrations. The role of these interactions in the circuits controlling the movements of the terminal appendages is discussed.Abbreviations AL antero-lateral - epsp excitatory postsynaptic potential - ipsp inhibitory postsynaptic potential - PL postero-lateral     

Down-regulation of human<Emphasis Type="Italic">NDR</Emphasis> gene in megakaryocytic differentiation of erythroleukemia K562 cells

To study the control of hematopoietic cell differentiation, a human negative differentiation regulator (NDR) gene was identified by the comparative analysis of differentially expressed genes in hemato-lymphoid tissues.NDR is expressed preferentially in the adult bone marrow, fetal liver and testis. Immunocytochemistry with anti-NDR antiserum showed the presence of NDR in human erythroleukemia K562 cell line and CD34⁺ cells sorted from the umbilical cord blood. When fused to the green fluorescent protein (GFP), NDR was directed to the nucleus of mouse 3T3 and K562 cells. Fusion protein with a deletion from residues 7 to 87 was detected in the cytoplasm. NDR appeared not to affect the proliferation of K562 cells when overly expressed. However, its expression was down-regulated during megakaryocytic differentiation of K562 cells induced by 12-O-tetradecanoylphorbol-13-acetate (TPA). Down-regulation of NDR correlated well with up-regulation of megakaryocytic markers, CD41 and CD61. Overexpression of the nuclear NDR-GFP in K562 cells inhibited the expression of CD41 and CD61 in megakaryocytic differentiation. Treatment of K562 cells with GF-109203X (GFX), an antagonist of the protein kinase C (PKC), blocked NDR down-regulation, up-regulated expression of CD41/CD61 and TPA-induced megakaryocytic differentiation. These results suggest a novel function of nuclear NDR protein in regulating hematopoietic cell development.     

Cryopreservation of umbilical cord blood: 1. Osmotically inactive volume,hydraulic conductivity and permeability of CD34(+) cells to dimethyl sulphoxide

Umbilical cord blood (UCB) is an accepted treatment for the reconstitution of bone marrow function following myeloablative treatment predominantly in children and juveniles. Current cryopreservation protocols use methods established for bone marrow and peripheral blood progenitors cells that have largely been developed empirically. Such protocols can result in losses of up to 50% of the nucleated cell population: losses unacceptable for cord blood. The design of optimal cryopreservation regimes requires the development of addition and elution protocols for the chosen cryoprotectant; protocols that minimise damaging osmotic transients. The biophysical parameters necessary to model the addition and elution of dimethyl sulphoxide to and from cord blood CD34(+) cells have been established. An electronic particle counting method was used to establish the volumetric response of CD34(+) cells to changes in osmolality of the suspending medium. The non-osmotic volume of the cell was 0.27 of the cells isotonic volume. The permeation kinetics of CD34(+) cells to water and dimethyl sulphoxide were investigated at two temperatures, +1.5 and +20 degrees C. Values for the hydraulic conductivity were 3.2 x 10(-8) and 2.8 x 10(-7)cm/atm/s, respectively. Values for the permeability of dimethyl sulphoxide at these temperatures were 4.2 x 10(-7) and 7.4 x 10(-6)cm/s, respectively. Clonogenic assays indicated that the ability of CD34(+) cells to grow and differentiate was significantly impaired outside the limits 0.6-4x isotonic. Based on the Boyle van't Hoff plot, the tolerable limits for cell volume excursion were therefore 45-140% of isotonic volume. The addition and elution of cryoprotectant was modelled using a two-parameter model. Current protocols for the addition of cryoprotectant based on exposure at +4 degrees C would require additional time for complete equilibration of the cryoprotectant. During the elution phase current protocols are likely to cause CD34(+) cells to exceed tolerable limits. The addition of a short holding period during elution reduces the likelihood of this occurring.