Impaired sperm function after spinal cord injury in the rat is associated with altered cyclic adenosine monophosphate signaling

Our previous observations of changes in the expression of cAMP-dependent genes and the cAMP-responsive element modulator (CREM) in rat testicular cells after spinal cord injury (SCI) implied abnormal cAMP signaling as one of the mechanisms underlying the effects of SCI on spermatogenesis. It was postulated that such effects might contribute to abnormal sperm function after SCI. In this study, we examined this possibility. In spinal cord-contused (SCC) and -transected (SCX) rats, impaired sperm motility was accompanied by an increase in sperm cAMP content. Treatment of SCX rats with exogenous testosterone or follicle-stimulating hormone resulted in a further decrease in sperm motility, whereas sperm cAMP either increased or remained unchanged. These effects differed from those in sham control rats that received identical treatments. Results of these experiments also demonstrated that impaired sperm motility in SCC and SCX rats was accompanied by decreases in sperm viability and mitochondrial potential, thus suggesting a possible link between these changes. We concluded that impaired sperm motility after SCI was associated with decreases in sperm viability and mitochondrial potential. These effects occurred in the face of elevated sperm cAMP content and changes in its regulation, suggesting that altered cAMP signaling events might contribute to impairment of sperm motility and perhaps other sperm functions after SCI.     

Alteration in articular cartilage of rat knee joints after spinal cord injury

OBJECTIVE: Mechanical forces are crucial for the maintenance of the morphologic and functional integrity of articular cartilage. The alteration of the articular cartilage after spinal cord injury (SCI) has been described in relation to a suppression of mechanical forces, since the joint is unloaded and restricted in movement. However, the morphological and biochemical characteristics of the cartilage after SCI are still poorly understood. We identified the localization of cartilage alterations after SCI and verified the influence of mechanical forces on the articular cartilage. METHOD: A total of 32 Wistar rats were used. Sixteen animals underwent an SCI and 16 animals served as control. The articular cartilage of the knee joint was assessed, respectively, at 4, 8, 10, and 12 weeks after intervention by histochemical, histomorphometric, immunohistochemical, and biochemical analyses. RESULTS: Cartilage thickness of spinal cord-injured knees decreased at the tibial and posterior femoral (FP) regions and increased at the anterior femoral (FA) region. Spinal cord injuries decreased the number of chondrocytes at the anterior regions and decreased the cartilage matrix staining only at the tibial regions. Immunolabeling to collagen type II was noted comparably in the superficial layer but noted weakly from the middle to deep layer. Collagen type I existed excessively at the cartilage surface and the pericellular regions. CONCLUSION: Cartilage alterations after SCI would not be explained by only a suppression of mechanical forces by unloading and immobilization, but there may be influences on the cartilage in addition to the change in mechanical forces.     

A cyclic adenosine 3',5'-monophosphate stimulates phospholipase Cgamma1-calcium signaling via the activation of tyrosine kinase in boar spermatozoa

The aim of this study was to reveal a downstream part of the intracellular signaling that is mediated by cyclic adenosine monophosphate (cAMP)-dependent tyrosine kinases, including spleen tyrosine (Y) kinase (SYK), in boar spermatozoa. Ejaculated spermatozoa were incubated with cBiMPS (a cell-permeable cAMP analog; 0.1 mM) at 38.5 degrees C for 180 minutes and then used for Western blot and indirect immunofluorescence. Incubation of spermatozoa with cBiMPS induced tyrosine phosphorylation at the linker region of SYK (which was essential to binding to phospholipase C [PLC]gamma1) in the connecting and principal pieces, but the tyrosine phosphorylation was abolished by the addition of H-89 (a protein kinase A [PKA] inhibitor; 0.01-0.1 mM). Moreover, the cAMP-dependent tyrosine phosphorylation was also induced at the key regulatory residue of PLCgamma1 in the same segments of spermatozoa, but it was inhibited by the addition of herbimycin A (a tyrosine kinase inhibitor; 5 microM). These results suggest that the sperm cAMP-dependent tyrosine kinases, including SYK, are linked to the activation of PLCgamma1. Indirect immunofluorescence clearly detected both inositol 1,4,5-trisphosphate (IP(3)) receptor and calreticulin in the connecting piece, indicating the presence of internal calcium store. Cell imaging with fluo-3/AM (a cell-permeable Ca(2+) indicator) showed that incubation of spermatozoa with cBiMPS increased intracellular free calcium in the middle piece, but that it was reduced by the addition of U-73122 (a PLC inhibitor; 0.02 mM). Based on our findings, we conclude that the connecting piece of boar spermatozoa possesses the PLCgamma1-IP(3) receptor-calcium signaling that is triggered by cAMP and mediated by PKA and herbimycin A-sensitive tyrosine kinases, including SYK.     

在大鼠切口疼痛模型中鞘内新斯的明对脊髓NO/cGMP信号转导系统的影响

目的 了解大鼠切口疼痛模型中鞘内（IT）新斯的明（NEO）对脊髓一氧化合酶（NOS）活性、一氧化氮（NO）产量和环鸟苷酸（cGMP）含量的影响。方法 雄性64只,随机分为四组,每组16只：假手术组（组I0、术前30minIT0．9％氯化钠20μl组（组Ⅱ）、术后30minITNEO10μg组（组Ⅲ）和术前30minITNEO10μg组（组Ⅳ）。按Brennan法制成切口疼痛模型,以累积疼痛评分确定疼痛行为。在术后2h断头取腰段脊髓,以分光光度法测定NOS活性、NO产量;放射免疫法测定cGMP含量／结果 组Ⅲ和组Ⅳ大鼠的累积评分均明显低于组Ⅱ（P＜0．01）。组Ⅱ的脊髓NOS活性、NO产量和cGMP含量均较组Ⅰ明显升高（P＜0．01）。组Ⅳ的脊髓NOS活性、NO产量和cGMP含量均较组Ⅰ明显升高（P＜0．01）。组Ⅳ的脊髓NOS活性、NO产量和cGMP含量均较组Ⅱ明显降低（P＜0．05或0．01）。而两用药组上述指标比较以及用药组与组I比较均无明显差别（P＞0．05）。结论 在大鼠切口疼痛模型中,术前IT NEO的抗伤害作用可能与NO／cGMP信号转导系统有关。     

Effect of parathyroid hormone and cyclic adenosine 3',5'-monophosphate on isotonic fluid reabsorption: polarity of proximal tubular cells.

Isotomic fluid reabsorption (JV) of rat renal proximal tubules was examined by the shrinking droplet method in combination with simulatneous perfusion of blood capillaries. Sensitivity of JV measurement was improved by using each punctured tubule for control measurements: 1) Parathyroid hormoen (PTH) on the contraluminal cell side reduced JV in a dose-response behavior. The maximal inhibition was achieved at a PTH concentration of 10(-5) M, the half maximal inhibition at a concentration of 3 X 10(-9) M. PTH on the luminal cell side had a small inhibitory effect. 2) Cyclic AMP inhibited JV preferentially when applied to the luminal cell side. On the luminal cell side, both cyclic AMP and dibutyryl cyclic AMP inhibited JV in a similar dose-dependent behavior. Concentrations of both nucleotides as low as 10(-10) M had a definite inhibitory effect. Tested at a high concentration, N6-butyryl cyclic AMP was almost as effective as cyclic AMP. Deoxy cyclic AMP, 5' AMP, cyclic guanosine monophosphate (cyclic GMP), dibutyryl cyclic GMP had no effect. ATP inhibited JV to a very small extent. 3) The reduction of JV after administration of PTH and dibutyryl cyclic AMP was not additive. The similar inhibitory effect of PTH at the contraluminal cell face and of cyclic AMP at the luminal cell face suggests the following sequence of events in the mediation of the action of PTH: 1) activation of adenylate cyclase by PTH in the contraluminal cell membrane, and 2) action of the generated cyclic AMP on the luminal cell membrane. The interaction of cyclic AMP and the luminal cell membrane is initiated at the luminal cell surface.     

大鼠脊髓损伤后巢蛋白在脊髓组织中的表达

目的探讨大鼠脊髓损伤后巢蛋白(nestin)的表达规律及其意义。方法30只Wister成年大鼠,随机分为正常对照组(A组)、损伤组(B组)。采用Allen打击模型(25g·cm),在T10段造成急性脊髓损伤,于损伤后1d、3d、1周、4周、8周进行取材,对距离损伤中心5mm处脊髓进行nestin免疫组化检测。应用图像分析软件进行nestin阳性区域面积侧算。结果A组脊髓室管膜细胞只可见极少数细胞胞浆内nestin表达,白质中几乎无表达。B组中nestin于损伤后24h表达于室管膜以及软膜,灰质和白质亦有少量表达,1周达到高峰(P<0.05),4周明显下降,8周时很少或几乎无表达。结论脊髓组织的许多部位可能存在具有分化和更新潜能的祖细胞,脊髓损伤后这些细胞被激活,在功能恢复中可能发挥着重要的作用。     

Effects of halothane on electrophysiologic properties and cyclic adenosine 3',5'-monophosphate content in isolated guinea pig hearts.

We studied the effects of halothane on the electrophysiologic and biochemical properties of both Langendorff perfused hearts and single ventricular myocytes isolated from guinea pigs. Isometric contractions of left ventricles in perfused hearts, elicited by atrial pacing, decreased to 14% of control after exposure to 2% halothane-equilibrated perfusate. Subsequently the slow inward Ca2+ current (ICa) was recorded in isolated myocytes with a whole cell voltage clamp technique. ICa, recorded in response to 100-ms depolarizations from -40 mV to 0 mV, was decreased by 2% halothane to 28.4% of control. Halothane-induced ICa depression did not exhibit use dependency. To define a possible site at which halothane acts, we measured the cyclic adenosine 3',5'-monophosphate (cAMP) content of single ventricular myocytes using a radioimmunoassay. Two percent halothane decreased myocardial cAMP content to 68.9% of control. Further addition of dibutyryl cAMP (10(-3) mol/L) partially reversed the depressed contractility during 2% halothane administration in perfused hearts. In conclusion, the present study demonstrated that the decrease of myocardial cAMP by halothane was due to a direct action, at least partly, and not to other factors such as catecholamines, and suggested that the decreases in contractility and ICa were induced possibly through the decrease in cellular cAMP.     

Inflammatory and apoptotic signaling after spinal cord injury

Central nervous system (CNS) destruction in spinal cord injury (SCI) is caused by a complex series of cellular and molecular events. Recent studies have concentrated on signaling by receptors in the tumor necrosis factor receptor (TNFR) superfamily that mediate diverse biological outcomes ranging from inflammation to apoptosis. From the perspective of basic science research, understanding how receptor signaling mediates these divergent responses is critical in clarifying events underlying irreversible cell injury in clinically relevant models of SCI. From a clinical perspective, this work also provides novel targets for the development of therapeutic agents that have the potential to protect the spinal cord from irreversible damage and promote functional recovery. In this review, we discuss how the formation of alternate signaling complexes and receptor membrane localization after SCI can influence life and death decisions of cells stimulated through two members of the TNFR superfamily, Fas/CD95 and TNFR1.     

Prostaglandins promote osteoclastlike cell formation by a mechanism involving cyclic adenosine 3',5'-monophosphate in mouse bone marrow cell cultures

We have developed a mouse bone marrow culture system in which multinucleated osteoclast (OC)-like cells are formed within 8 days. Using this culture system, we examined the effect of prostaglandins (PGs), potent bone-resorbing agents, on OC-like cell formation. Four PGs (PGE1 and PGE2 at 10(-8)-10(-5) M, 6-keto-PGF1 alpha at 10(-5) M, and PGF2 alpha at 10(-6)-10(-5) M) significantly stimulated the formation of OC-like cells. The potency of the PGs in inducing OC-like cell formation was the highest in PGE1 and PGE2, followed by PGF2 alpha and 6-keto-PGF1 alpha in that order, and the order was highly correlated with the order of the potency in increasing the production of cyclic adenosine 3',5'-monophosphate (cAMP) in bone marrow cells. Addition of dibutyryl-cAMP also induced OC-like cell formation. Moreover, isobutylmethylxanthine (IBMX), a potent inhibitor of phosphodiesterase, potentiated the OC-like cell formation induced by PGE2, whereas salmon calcitonin greatly inhibited it. Calcitonin induced cAMP production in cultures treated with PGE2, but not in cultures with vehicle. When bone marrow mononuclear cells were cultured on dentine slices in the presence of PGE2, multinucleated OC-like cells were similarly formed and they resorbed calcified dentine, resulting in so-called Howship's lacunae. These results suggest that PGs stimulate resorption of calcified tissues by promoting osteoclast formation. The activity of PGs in inducing OC-like cell formation is considered mediated mainly by a mechanism involving cAMP.     

Viability and protein phosphorylation patterns of boar spermatozoa agglutinated by treatment with a cell-permeable cyclic adenosine 3',5'-monophosphate analog

Boar spermatozoa become agglutinated with one another at the head when their intracellular cyclic adenosine 3',5'-monophosphate (cAMP)-signaling cascades are activated in the head. The aim of the present study is to examine viability and protein phosphorylation patterns of cAMP-dependently agglutinated boar spermatozoa. Ejaculated spermatozoa were washed and then incubated in a modified Krebs-Ringer HEPES medium containing polyvinyl alcohol (mKRH-PVA) plus 0.1 mM Sp-5,6-dichloro-1-beta-D-ribofuranosyl-benzimidazole-3',5'-monophosphorothioate (cBiMPS, a cell-permeable cAMP analog) at 38.5 degrees C up to 180 minutes. Aliquots of the sperm suspensions were recovered after various incubation periods and then used to examine the state of agglutination, the viability by SYBR14-PI staining and motility assay, and the state of protein phosphorylation by Western blotting and indirect immunofluorescence. In the control samples incubated without cBiMPS for 180 minutes, less than 30% of the total spermatozoa were agglutinated with one another at the heads, and more than 70% of the agglutinated spermatozoa were propidium iodide (PI)-positive (dead). However, the incubation with cBiMPS rapidly increased the percentages of head-to-head agglutinated spermatozoa to approximately 60% within 30 minutes, but did not significantly change them thereafter. In the samples incubated with cBiMPS for 180 minutes, moreover, the percentages of PI-positive cells of the agglutinated spermatozoa (approximately 30%) were significantly lower than those obtained in the control samples (more than 70%). This result was supported by the observation that the percentages of motile cells of the agglutinated spermatozoa were much higher in the samples incubated with cBiMPS for 180 minutes than in the control samples incubated without cBiMPS. As revealed by Western blotting and indirect immunofluorescence, cBiMPS-induced serine/threonine phosphorylation of the proteins (eg, >220 kd, 220 kd, 180 kd, 84 kd, and 54 kd) appeared mainly in the connecting and principal pieces of both agglutinated and free spermatozoa within 30 minutes, and additional phosphorylation occurred in the middle piece later than 30 minutes. Moreover, tyrosine phosphorylation of the proteins (eg, >220 kd, 190 kd, 93 kd, 59 kd, 54 kd, and 32 kd) was induced intensely in the connecting and principal pieces and moderately in the middle piece of almost one half of the agglutinated spermatozoa after incubation with cBiMPS for more than 30 minutes, but rarely in those of the free spermatozoa. These findings are consistent with the following suggestions: activation of the cAMP-signaling cascades leads to rapid (within 30 minutes) head-to-head agglutination in live spermatozoa; rapid (within 30 minutes) protein serine/threonine phosphorylation in the connecting and principal pieces of both cAMP-dependently agglutinated and free spermatozoa and subsequent (later than 30 minutes) phosphorylation in the middle piece of them; and slow (later than 30 minutes) protein tyrosine phosphorylation in the connecting, middle, and principal pieces of the cAMP-dependently agglutinated spermatozoa. Based on these suggestions, we conclude that many of cAMP-dependently agglutinated spermatozoa are live cells in which cAMP-signaling cascades leading to protein serine/threonine and tyrosine phosphorylation are activated in the whole flagellum.     

Deuterated halothane--anesthetic potency, anticonvulsant activity, and effect on cerebellar cyclic guanosine 3',5'-monophosphate

The effect of substituting deuterium for hydrogen in the halothane molecule on anesthetic potency, motor activity, and cerebellar cyclic guanosine 3',5'-monophosphate (cGMP) content was studied in mice. The concentration of halothane required to abolish the righting reflex in 50% of the mice (ED50RR) was chosen as index of anesthetic potency; cerebellar control of motor activity was evaluated by the incidence of isoniazid-induced convulsions. The ED50RR for deuterated (D)-halothane was similar to that of halothane (0.87 +/- 0.04 and 0.88 +/- 0.03 vol%, respectively). Both D-halothane and halothane (0.15-0.90 vol%) protected the mice against isoniazid-induced convulsions and decreased cerebellar cGMP content in a dose-dependent manner. D-halothane and halothane were equipotent on both parameters. Thus deuteration did not alter the anesthetic potency, the anticonvulsant activity, or the effect on cerebellar cGMP content of the anesthetic. Furthermore, the reactivity of the C-H bond is probably not critical for these actions of halothane.     

Transplantation of adult rat spinal cord stem/progenitor cells for spinal cord injury

Stem/progenitor cells derived from the ependymal region of the spinal cord have the ability to self-renew and are multipotential for neurons and glia. These cells may have the ability to regenerate the injured mammalian spinal cord as they do in some lower vertebrates. However, the optimal conditions for transplantation and the fate of transplanted cells are not fully known. In the current study, spinal cord stem/progenitor cells were cultured from adult male rats expressing enhanced green fluorescent protein (eGFP). Neurospheres were transplanted at the time of clip compression injury (35-g force) into the injury site, or 1 mm rostral and caudal to the injury site. Neurospheres were also transplanted into a subacute model (day 9 after injury) and a chronic model (day 28 after injury). Functional recovery was also studied in an acute injury model with weekly locomotor testing over a 16-week period. A significant increase in cell survival at 7 days was seen in rats receiving rostral and caudal injections as compared to injection directly into the site of injury. A significant increase in cell survival was also seen in rats receiving subacute transplants at 9 days after injury. Transplanted cells differentiated primarily into astrocytes (31.2%) and oligodendrocytes (50.3%), and a small number of neurons (1%). No improvement was seen in the Basso, Beattie and Bresnahan (BBB) locomotor rating scale after acute transplantation as compared with injury only, although surviving transplanted cells were identified that had migrated across the injury site from the rostral and caudal injection sites.     

大鼠脊髓损伤后Wnt信号分子的表达变化

目的:探讨大鼠脊髓损伤后不同时期Wnt信号分子Wnt-1、β-连锁蛋白(β-catenin)及糖原合成酶激酶-3β(GSK-3β)在脊髓损伤局部的表达情况.方法:50只成年雌性SD大鼠随机均分为对照组和实验组,麻醉下手术显露T9～T11椎板,切除T10全椎板,实验组大鼠用NYU打击器以10g×5cm的打击能量致伤T10脊髓,对照组只行全椎板切除,不致伤脊髓.分别于术后1d、3d、5d、7d、14d每组各取5只大鼠,取以损伤区为中心共15mm范围内(对照组取相应部位)脊髓组织,提取总RNA,采用半定量RT-PCR的方法检测脊髓组织中Wnt-1、β-catenin及GSK-3β的mRNA表达量.结果:脊髓损伤后1d和3d时Wnt-1和β-catenin出现高表达,5d后其表达逐渐减弱,14d左右其表达基本恢复到正常水平,而在脊髓损伤后1d和3d时GSK-3β呈低表达,5d后其表达逐渐增强,各时间点之间差异有显著性(P<0.05).对照组中Wnt-1和β-catenin及GSK-3β均呈低表达,各时间点表达无显著差异(P<0.05).结论:大鼠脊髓损伤后损伤局部脊髓组织中Wnt-1,β-catenin及GSK-3β的表达发生变化,提示Wnt信号在脊髓损伤后的早期被激活,其可能与脊髓损伤后的修复再生有关.     

Prospective analysis of intraoperative and postoperative urinary cyclic adenosine 3',5'-monophosphate levels to predict outcome of patients undergoing reoperations for primary hyperparathyroidism

Twenty-five consecutive patients with either persistent or recurrent symptomatic primary hyperparathyroidism after 36 prior operations were prospectively studied to compare the usefulness of intraoperative measurement of urinary cyclic adenosine monophosphate (UcAMP) levels with standard surgical procedures to predict outcome during tedious parathyroid reoperations. The criterion based on UcAMP to predict successful outcome was a 50% decline in intraoperative UcAMP levels from the median baseline level after removal of abnormal parathyroid tissue. Standard surgical criteria were resection of four abnormal glands for hyperplasia and resection of one abnormal gland and biopsy examination of one normal gland for adenoma. In 15 patients (60%) surgery was terminated on the basis of UcAMP criterion. In one patient elevated UcAMP levels never changed and correctly predicted unsuccessful surgery. In nine patients surgery was terminated on the basis of surgical criteria, and each of these patients had a successful outcome. Operative UcAMP levels dropped after the completion of the procedure in six of these latter nine patients. Three patients did not show a significant decrease in UcAMP levels despite successful surgery, and one patient displayed an early false-positive decrease in UcAMP level. The intraoperative UcAMP criterion accurately predicted outcome in 21 of 25 patients (84%). Sensitivity of the UcAMP criterion was 87% and specificity was 50%. The results indicate that by either method a reliable prediction of the outcome of reoperative parathyroid surgery may be made. Intraoperative determination of UcAMP levels will allow successful termination of the reoperation in some patients before operative identification of adequate parathyroid tissue necessary to confidently establish the pathologic diagnosis.     

Axonal remyelination by cord blood stem cells after spinal cord injury

Human umbilical cord blood stem cells (hUCB) hold great promise for therapeutic repair after spinal cord injury (SCI). Here, we present our preliminary investigations on axonal remyelination of injured spinal cord by transplanted hUCB. Adult male rats were subjected to moderate SCI using NYU Impactor, and hUCB were grafted into the site of injury one week after SCI. Immunohistochemical data provides evidence of differentiation of hUCB into several neural phenotypes including neurons, oligodendrocytes and astrocytes. Ultrastructural analysis of axons reveals that hUCB form morphologically normal appearing myelin sheaths around axons in the injured areas of spinal cord. Colocalization studies prove that oligodendrocytes derived from hUCB secrete neurotrophic hormones neurotrophin-3 (NT3) and brain-derived neurotrophic factor (BDNF). Cord blood stem cells aid in the synthesis of myelin basic protein (MBP) and proteolipid protein (PLP) of myelin in the injured areas, thereby facilitating the process of remyelination. Elevated levels of mRNA expression were observed for NT3, BDNF, MBP and PLP in hUCB-treated rats as revealed by fluorescent in situ hybridization (FISH) analysis. Recovery of hind limb locomotor function was also significantly enhanced in the hUCB-treated rats based on Basso-Beattie-Bresnahan (BBB) scores assessed 14 days after transplantation. These findings demonstrate that hUCB, when transplanted into the spinal cord 7 days after weight-drop injury, survive for at least 2 weeks, differentiate into oligodendrocytes and neurons, and enable improved locomotor function. Therefore, hUCB facilitate functional recovery after moderate SCI and may prove to be a useful therapeutic strategy to repair the injured spinal cord.