Type-1 cannabinoid receptor expression in the frog, Rana esculenta, tissues: a possible involvement in the regulation of testicular activity

Endogenous cannabinoids and type-1 cannabinoid receptor (CB1) are widely produced and distributed in the central nervous system (CNS) and peripheral nerves in mammals. In addition, the detection of endocannabinoids and corresponding receptors in non nervous peripheral tissues indicates an involvement of the system in the control of a wide range of physiological activities, including reproduction. Recently, the existence of CB1 was also observed in lower vertebrates and in urochordate suggesting that the endocannabinoid system is phylogenetically conserved. Using RT-PCR, CB1 mRNA expression profiles were characterized in a wide range of tissues of the anuran amphibian, the frog, Rana esculenta. Besides a strong expression in the CNS, CB1 was also present in testis, kidney, liver, ovary, muscle, heart, spleen, and pituitary. The CB1 expression pattern has been characterized in both testis and CNS during the annual sexual cycle. In testis, CB1 is poorly expressed during the winter stasis of the spermatogenesis rising during the breeding season and resumption period. An expression profile mismatching to that observed in testis was detected in whole-brain preparations during the sexual cycle; in particular in the diencephalon, the encephalic area mainly involved in the control of reproductive functions. Furthermore, fluctuations inside isolated encephalic areas and spinal cord were observed all over the reproductive cycle. In conclusion, CB1 receptor is expressed in R. esculenta CNS and testis. As far as the gonad it concerns, our results suggest the involvement of the endocannabinoids in the control of reproductive function.     

人睾丸与脑基因表达谱的相似性

人类的物种形成与进化问题一直是研究的一个焦点。近年来,对于人和灵长类以及果蝇等其他一些动物多种组织基因表达谱的研究表明,在人的进化过程中脑基因表达的改变最为显著,并且脑中许多基因的表达呈显著上调。信息学分析显示,在多种组织当中,人的脑与睾丸可能存在最为相似的基因表达谱。这些结果提示睾丸可能与脑类似,也在人的物种形成和进化历程中起着重要作用。本文对人睾丸和脑基因表达谱的研究进行了回顾,并提出了该研究方向今后的一些研究设想。     

Altered chemokine expression in the spinal cord and brain contributes to differential interleukin-1beta-induced neutrophil recruitment

The pattern of neutrophil recruitment that accompanies inflammation in the CNS depends on the site of injury and the stage of development. The adult brain parenchyma is refractory to neutrophil recruitment and associated damage as compared to the spinal cord or juvenile brain. Using quantitative Taqman RT-PCR and enzyme-liked immunosorbent assay (ELISA), we compared mRNA and protein expression of the rat neutrophil chemoattractant chemokines (CINC) in spinal cord and brain of adult and juvenile rats to identify possible association with the observed differences in neutrophil recruitment. Interleukin-1beta (IL-1beta) injection resulted in up-regulated chemokine expression in both brain and spinal cord. CINC-3 mRNA was elevated above CINC-1 and CINC-2alpha, with expression levels for each higher in spinal cord than in brain. By ELISA, IL-1beta induced greater CINC-1 and CINC-2alpha expression compared to CINC-3, with higher protein levels in spinal cord than in brain. In the juvenile brain, significantly higher levels of CINC-2alpha protein were observed in response to IL-1beta injection than in the adult brain following an equivalent challenge. Correspondingly, neutrophil recruitment was observed in the juvenile brain and adult spinal cord, but not in the adult brain. No expression of CINC-2beta mRNA was detected. Thus differential chemokine induction may contribute to variations in neutrophil recruitment in during development and between the different CNS compartments.     

Neuregulin-1-beta1 enters brain and spinal cord by receptor-mediated transport

Proteins of the neuregulin (NRG) family play important regulatory roles in neuronal survival and synaptic activity. NRG-1-beta1 has particular potential as a therapeutic agent because it enhances myelination of neurites in spinal cord explants. In this study, we determined the permeation of NRG-1-beta1 across the blood-brain and blood-spinal cord barriers (BBB and BSCB respectively). Intact radioactively labeled NRG-1-beta1 had a saturable and relatively rapid influx rate from blood to the CNS in mice. Capillary depletion studies showed that NRG-1-beta1 entered the parenchyma of the brain and spinal cord rather than being trapped in the capillaries that compose the BBB. The possible mechanism of receptor-mediated transport was shown by the ability of antibodies to erbB3 and erbB4 receptors to inhibit the influx. Lipophilicity, less important for such saturable transport mechanisms, was measured by the octanol : buffer partition coefficient and found to be low. The results indicate that NRG-1-beta1 enters spinal cord and brain by a saturable receptor-mediated mechanism, which provides the opportunity for possible therapeutic manipulation at the BBB level.     

Isolated spinal cord contusion in rats induces chronic brain neuroinflammation,neurodegeneration, and cognitive impairment

Cognitive dysfunction has been reported in patients with spinal cord injury (SCI), but it has been questioned whether such changes may reflect concurrent head injury, and the issue has not been addressed mechanistically or in a well-controlled experimental model. Our recent rodent studies examining SCI-induced hyperesthesia revealed neuroinflammatory changes not only in supratentorial pain-regulatory sites, but also in other brain regions, suggesting that additional brain functions may be impacted following SCI. Here we examined effects of isolated thoracic SCI in rats on cognition, brain inflammation, and neurodegeneration. We show for the first time that SCI causes widespread microglial activation in the brain, with increased expression of markers for activated microglia/macrophages, including translocator protein and chemokine ligand 21 (C–C motif). Stereological analysis demonstrated significant neuronal loss in the cortex, thalamus, and hippocampus. SCI caused chronic impairment in spatial, retention, contextual, and fear-related emotional memory—evidenced by poor performance in the Morris water maze, novel objective recognition, and passive avoidance tests. Based on our prior work implicating cell cycle activation (CCA) in chronic neuroinflammation after SCI or traumatic brain injury, we evaluated whether CCA contributed to the observed changes. Increased expression of cell cycle-related genes and proteins was found in hippocampus and cortex after SCI. Posttraumatic brain inflammation, neuronal loss, and cognitive changes were attenuated by systemic post-injury administration of a selective cyclin-dependent kinase inhibitor. These studies demonstrate that chronic brain neurodegeneration occurs after isolated SCI, likely related to sustained microglial activation mediated by cell cycle activation.     

Sciatic nerve transection decrease substance P immunoreactivity in the lumbosacral spinal cord of the frog (Rana catesbeiana)

Using immunohistochemistry and optical densitometry, substance P (SP) was investigated in the lumbar spinal cord of the frog Rana catesbeiana after sciatic nerve transection. In control animals, there was a high density of SP fibers in the Lissauer's tract and in the mediolateral band of the dorsal gray matter. Other SP immunoreactive fibers were observed in the dorsal part of the lateral funiculus and in the ventral horn. No SP label was found in any cell bodies. After axotomy, SP immunoreactive fibers decreased in the Lissauer's tract on the same side of the lesion. The other regions remained labeled. The changes were observed at 3 days following axonal injury and persisted at 5, 8 and 15 days. At 20 days, there was no significant difference between the axotomized side and the control one, thus indicating a recovery of the SP expression. These results indicate that the frog may be used as a model to study the effects of peripheral axotomy, contributing to elucidate the SP actions in the pain neuropath.     

Utilization of ketone bodies by chick brain and spinal cord during embryonic and postnatal development

Lipid synthesis from acetoacetate and 3-hydroxybutyrate was studied in chick embryo from 15 to 21 days and in chick neonate from 1 to 21 days. Embryonic spinal cord showed higher ability than brain to incorporate acetoacetate into total lipids, although a sharp decrease was found at hatching. 3-Hydroxybutyrate incorporation into total lipids was also higher in spinal cord than in brain, especially during the embryonic period. Phospholipids were the main lipids formed in both tissues from both precursors. An appreciable percentage of radioactivity was also recovered as free cholesterol, especially during the embryonic phase. The developmental patterns of amino acid synthesis from acetoacetate and 3-hydroxybutyrate were similar in both tissues: a clear increase after hatching was followed by a decrease at day 4 of neonatal life. Acetoacetate was a better substrate for amino acid synthesis than 3-hydroxybutyrate during the embryonic development in both tissues. Oxidation of both precursors to CO₂ strongly decreased between 15 and 21 days of embryonic development both in brain and spinal cord.     

神经激肽-1(NK-1)受体介导福尔马林诱发的大鼠脊髓c-fos表达

本文用Ｆｏｓ作为背角伤害性反应神经元活动的一个标志物,结合免疫细胞化学和神经药理学方法,观察了速激肽受体拮抗剂对福尔马林诱发的脊髓ｃ－ｆｏｓ原癌基因表达的影响。一侧大鼠后肢跖部皮下注射福尔马林,仅在同侧脊髓背角有ｃ－ｆｏｓ表达。Ｆｏｓ阳性神经元最密集分布于Ｉ层和Ⅱ层背侧的内侧部,中等量分布于Ⅳ层和Ｖ型,少量定位于Ⅱ层腹侧部、Ⅲ、Ⅵ和Ⅹ层。若预先在一侧大鼠后肢跖部皮下注射福尔马林前,鞘内给予神经激肽     

甲醛炎性痛对大鼠脊髓HO-1表达的影响

目的:观察足底注射甲醛引起的外周组织炎性疼痛是否可诱导大鼠脊髓血红素氧合酶-1(HO-1)表达发生改变以及变化的时程特征。方法:健康雄性SD大鼠随机分为7组(n=6):对照组(control组)、甲醛6 h组(F6 h组)、甲醛12 h(F12 h组)、甲醛1 d组(F1 d组)、甲醛2 d组(F2 d组)、甲醛3 d组(F3 d组)和甲醛7 d组(F7 d组)。采用足底注射甲醛溶液复制炎性痛模型,采用免疫组织化学方法检测左、右两侧脊髓后角以及中央管周围灰质HO-1蛋白的表达。结果:Control组大鼠HO-1免疫反应阳性细胞在脊髓后角及中央管周围灰质仅有少量分布,且这些细胞染色较浅。足底注射甲醛后6 h,L5节段双侧脊髓后角和中央管周围灰质HO-1免疫反应阳性细胞数目即有所增多,足底注射甲醛后12 h时,双侧脊髓后角和中央管周围灰质HO-1免疫反应阳性细胞数目进一步增多,阳性细胞染色明显加深,1 d时阳性细胞数目和染色深度均达到高峰,7 d时仍高于control组水平。各时间点双侧脊髓后角比较,阳性细胞数目和阳性细胞染色深度均无明显差异。结论:大鼠足底注射甲醛引起的炎性痛可诱导双侧脊髓后角和中央管周围灰质HO-1表达增多,以注射甲醛后1 d时增多最为明显。     

Early gene expression during natural spinal cord regeneration in the salamander Ambystoma mexicanum

In contrast to mammals, salamanders have a remarkable ability to regenerate their spinal cord and recover full movement and function after tail amputation. To identify genes that may be associated with this greater regenerative ability, we designed an oligonucleotide microarray and profiled early gene expression during natural spinal cord regeneration in Ambystoma mexicanum. We sampled tissue at five early time points after tail amputation and identified genes that registered significant changes in mRNA abundance during the first 7 days of regeneration. A list of 1036 statistically significant genes was identified. Additional statistical and fold change criteria were applied to identify a smaller list of 360 genes that were used to describe predominant expression patterns and gene functions. Our results show that a diverse injury response is activated in concert with extracellular matrix remodeling mechanisms during the early acute phase of natural spinal cord regeneration. We also report gene expression similarities and differences between our study and studies that have profiled gene expression after spinal cord injury in rat. Our study illustrates the utility of a salamander model for identifying genes and gene functions that may enhance regenerative ability in mammals.     

Fra1 activity in the frog, Rana esculenta, testis: a new potential role in sperm transport

Using an anti-Fos family member antibody, we have previously described in Rana esculenta testis the presence of a nuclear, 43 kDa protein that we hypothesized to be Fra1. With the assistance of an antibody against Fra1 that does not cross-react with other Fos family members, here we report data on Fra1 expression, localization, and putative activity in Rana esculenta testis during its annual reproductive cycle. Western blot analysis confirms that the nuclear, 43 kDa protein is Fra1. Immunocytochemistry validates the Western blot results and shows cytoplasmic and nuclear immunostaining of Fra1 in peritubular myoid cells, efferent ducts, and blood vessels. We report for the first time in a vertebrate, experimental evidence showing that the expression of Fra1 is related to peritubular myoid cells during sperm transport from the tubular compartment to efferent ducts.     

The annual cycle of activity of the subfornical organ in the green frog Rana esculenta

Abstract

The nuclear volume in cells of the subfornical organ of Rana esculenta shows a cyclic annual size change. It is pronounced both in the so‐called Gomori‐positive cells and the ependymal covering of the organ. There appears a maximum in April in the epen‐dyma and in May in the Gomori‐positive cells. The amount of neurosecretory material present in the subependymal layer also shows a maximum in April.