Some characteristics of muscle-conditioned media with excitatory and inhibitory influences on neurite outgrowth from early neural tube explants

This study examined the growth of basal plate explants from 40-hr chick embryos containing the trigeminal motor nucleus in the presence of target muscle-conditioned medium (MCM) from day-4, day-10, and day-18 embryos. We had previously found that MCM derived from the 4-day target tissue enhanced neurite outgrowth from these explants, while target MCM from 10- and 18-day embryos inhibited it. For the present study, target MCM was treated with trypsin to assess the nature of the active fractions, or pre-incubated in polyornithine-coated dishes to determine the possible presence and relative contributions of substrate-binding vs. soluble components. Both trypsinization and pre-incubation abolished the outgrowth-enhancing potential of the 4-day MCM, indicating a protein or polypeptide substrate-binding active fraction, with no appreciable active soluble component. When the 10-day and 18-day target MCM were trypsinized, the inhibitory influence was reversed, and neurite outgrowth was enhanced. Similarly, when the MCM from these embryos was pre-incubated over polyornithine, the inhibitory influence was lost and was replaced by a stimulatory one. These results suggest that the 10- and 18-day MCM contain an active inhibitory fraction that is a protein or polypeptide, and which binds to a positively charged substrate. When this inhibitory fraction is inactivated or depleted, an excitatory soluble fraction is revealed. Such a dual nature in MCM has not been seen previously and may be expressed only when the media are assayed with very early neural tissue.     

Comparative responsiveness of early chick embryo brainstem and spinal cord neurons to target-conditioned medium

Neurons from the early trigeminal (V) region of the basal plate and from the early ventral spinal cord of chick embryos were dissociated. Their responsiveness to muscle-conditioned medium (MCM) derived from age-matched appropriate and inappropriate target was compared. The V neurons responded to appropriate but not inappropriate MCM by enhanced survival and increased neurite production. Conversely, there were no differences in these measures in spinal cord neurons cultured in control medium and in MCM derived either from appropriate or inappropriate (trigeminal) target. The differences in specificity expressed by these two early populations is discussed.     

Endogenous and exogenous factors support neuronal survival and choline acetyltransferase activity in embryonic spinal cord cultures

Dissociated 4-day (stage 23) chick embryo lumbar cord cells were cultured at low or high cell densities for 1 or 5 days in the presence or absence of added spinal neuronotrophic factor (supplied as RN22 Schwannoma conditioned medium, RCM). In low density, 1-day cultures neuronal survival was dependent on added RCM whereas by 5 days no neurons survived, even in the presence of RCM. In high density 1-day cultures a substantial neuronal population could survive even without added RCM and a large proportion of this neuronal population would survive for 5 days. When conditioned media from high density lumbar cord cultures was supplied to low density unsupplemented cultures, a similar level of 5-day neuronal survival resulted. However, no neurons survived in RCM-supplemented 5-day high density cultures, indicating the presence in RCM of a material toxic for the neurons. Both the RCM and the high density lumbar culture-conditioned medium supported considerable choline acetyltransferase activity indicating the presence within these cultures of motoneurons.     

Specific responsiveness of chick trigeminal motor nucleus explants to target-conditioned media

Explants of the neural tube from stage 11 chick embryos containing the metencephalic trigeminal (V) motor nucleus were cultured in standard control medium, in medium conditioned by appropriate target musculature (the mandibular process of the first visceral arch that gives rise to jaw musculature innervated by motor V) or in medium conditioned by inappropriate target musculature (rostral limb bud tissue). The appropriate and inappropriate muscle tissues were of the same developmental stage (stage 22) and were in similar states of differentiation. At this point in vivo, both are just beginning to be innervated. The neuritic outgrowth from the explants was quantified after 6 days in vitro. While explants from all three groups appeared healthy and exhibited some neuritic outgrowth, the density and complexity of this growth was significantly greater in the group cultured with the appropriate (jaw) muscle-conditioned medium. Growth in this group significantly surpassed that of both the control and the inappropriate muscle-conditioned medium group did not differ from the control group. These results demonstrate a specific responsiveness of the trigeminal motor nucleus population to its appropriate target tissue. Since relatively small amounts of the muscle-conditioned medium were used with each explant, it is suggested that there is a high degree of sensitivity of this population to factors present in their target at the time innervation would normally be occurring. It is hypothesized that such selective responsiveness may play a role in guiding or sustaining growth during normal neurogenesis.     

Sources of trophic factors that induce limb regeneration and prevent amputation-induced neuronal death

Segments of 2-, 4-, 6- and 8-day neural tube, or of 15-day peripheral nerve were implanted longitudinally into limb stumps of 4-day chick embryos whose right-wing buds were amputated at the future elbow region. Stumps of amputated limbs (ALs) implanted with 7-day heart or without implant served as controls. Effects of progressively older neural tube implants (NTIs) upon ALs and host spinal cord neurons were analyzed by area measurements of the peripheral limb field (PLF) and NTI and by cell counts of the host lateral motor column (LMC). Nine days postamputation, 2- and 4-day NTIs contained many neurons and induced epimorphic regeneration in more than one-fourth of the embryos. Six-day NTIs contained few neurons and induced only tissue regeneration. Eight-day NTIs and peripheral nerve containing only non-neuronal cells were as ineffective as controls in stimulating regeneration, although peripheral nerve did cause a significant increase in the peripheral field. The NTIs of all ages and implants of peripheral nerve were equally effective in protecting LMC neurons from amputation-induced cell death in the host spinal cord. The results may indicate that neurons of the implant induce limb regeneration and non-neuronal cells of the implant protect against LMC neuronal death.     

Morphological response of extending spinal neuritic growth cones to peripheral target tissue.

Nerve fibers extend from spinal cord explants of larval frog in an enhanced and directed manner when cocultured with limb mesenchyme target tissue. In order to gain a better understanding of the events involved in target directed neurite extension, a detailed examination of the nerve growth cone was undertaken. The growth cones of spinal neurites that had elongated in the presence or absence of target tissue were examined by light and electron microscopy. Scanning electron microscopy revealed that growth cones of cord+limb cultures were elaborate in form with numerous and long filopodia, while those cultured in the absence of the target tissue appeared relatively simple with few, short filopodia. A morphological parallel existed between those growth cones that were cultured without the target and those in cord+limb cultures but which grew from the side of the cord explant away from the mesenchyme tissue. When examined with the transmission electron microscope, growth cones under target influence were organelle-rich in contrast to target-deprived growth cones, which lacked the extensive array of vesicles, endoplasmic reticulum, and filaments. When the attachment substratum of polylysine was substituted by collagen, the dramatic differences in growth cones were not realized, although enhanced, oriented growth still occurred in the presence of limb target tissue. It appears that growth cone morphology is a dynamic reflection of the growth effects elicited by a target tissue factor that in turn may be mediated by the nature of the extracellular environment.     

Basal lamina and superfast myosin expression in regenerating cat jaw muscle

We investigated the possible role of extracellular matrix in specifying the expression of superfast myosin during cat jaw muscle regeneration. Equal proportions of muscle tissue from jaw and limb were minced together after killing cellular elements from one source. We allowed the mince to regenerate in the bed of a fast limb muscle. Regenerates were analyzed immunocytochemically at 71 to 294 days after operation. Fibers in control regenerates containing live cells from both sources expressed fast, superfast or slow myosins, or a mixture of these myosins. In regenerates containing only one type of live cells, we detected only myosins appropriate to the live cells. Our results suggest that during regeneration the original extracellular matrix of jaw-closing or limb muscle is unable to specify the expression of superfast or fast myosins, respectively; they point to the cellular elements, probably the satellite cells, as determinants of muscle specificity during regeneration.     

Enhanced release of von Willebrand factor by human endothelial cells in culture in the presence of phorbol myristate acetate and interleukin 1

Human umbilical vein endothelial cells (EC) were cultured in the presence of 4 beta-phorbol 12-myristate 13-acetate (PMA), interleukin 1 (IL-1) and interleukin 2 (IL-2), and secretion of von Willebrand factor activity (vWF, Ristocetin Co-factor) and von Willebrand factor antigen (vWFAg) measured at intervals. The levels of both vWF and vWFAg in cultures containing IL-1 were significantly higher than those in control cultures after longer than 5-6 days growth. Similarly, the levels of vWF and vWFAg were significantly increased in cultures containing PMA, but in these instances the rate of cell growth appeared to be enhanced, and confluence was observed after 6 days compared to 9-10 days in control cultures. vWF and vWFAg also increased significantly in the supernatant of confluent control EC incubated further with IL-1. Moreover, the immuno-fluorescence pattern of vWFAg in these treated cells was markedly less granular than that of control cells. Immuno-fluorescence of PMA-treated cells suggested that vWFAg might be less granular than in control EC but the mean levels of vWF and vWFAg in the supernatant of cells incubated with PMA were not significantly different from control values. The results of all assays in the presence of IL-2 were not significantly different from those of control cells. In all instances no morphological evidence of endothelial injury was observed, and more than 90% of cells remained viable at the termination of cultures. The results indicated that the synthesis and release of vWF were increased in the presence of PMA, and secretion of vWF was stimulated by IL-1.(ABSTRACT TRUNCATED AT 250 WORDS)     

Estrogen affects levels of Bcl-2 protein and mRNA in medial amygdala of ovariectomized rats

The survival factor Bcl-2 is a cyclic AMP response element-binding protein (CREB) gene product implicated in mediating some of estrogen's effects on neuroprotection. Previously, we showed an effect of estradiol benzoate (E) on numbers of neuron-specific protein (NeuN)- and phosphorylated CREB (pCREB)-positive cells in medial (MeA), but not central (CeA), amygdala of ovariectomized rats. To determine whether these effects are accompanied by an E-induced increase in Bcl-2, we examined the effects of E on levels of Bcl-2 protein and mRNA in MeA and CeA of ovariectomized rats treated with E regimens resulting in moderate (2.5 microg E for 4 or 14 days) or high (10 microg E for 14 days) plasma estradiol levels. As a physiological control, we showed that all E treatments increased uterine wet weight relative to vehicle; 10 microg E for 14 days also increased uterine weight compared with that seen with lower E levels. Western blot analysis revealed that all E groups displayed an increase in uterine Bcl-2 protein levels compared with vehicle. We found that 2.5 microg and 10 microg E for 14 days increased levels of Bcl-2 gold immunolabeling compared with vehicle and 2.5 microg E for 4 days in MeA, but not CeA. We measured Bcl-2 mRNA levels in vehicle and 2.5 microg E-treated 14-day groups. There was a significant increase in Bcl-2 mRNA levels in MeA, but not CeA, of E-treated ovariectomized rats compared with vehicle controls. The E-induced increase in protein and mRNA levels of Bcl-2 in MeA may be important for neuroprotection in this region.     

Development of NADPH-diaphorase activity in the central nervous system of the cichlid fish, Tilapia mariae

The distribution of nicotinamide adenine dinucleotide phosphate (NADPH)-diaphorase activity was studied in the cichlid fish Tilapia mariae during ontogenesis by the histochemical reaction of NADPH-diaphorase that indicates, in aldehyde-fixed tissue, the presence of nitric oxide synthase, which is the enzyme responsible for nitric oxide production. The first appearance of NADPH-diaphorase-positive neurons has a striking bilateral symmetry and occurs 20 h after fertilization (stage 8) in the olfactory placodes and in the neural tube where two clusters of positive neurons were seen in the diencephalon and in the rhombomere r4 of the hindbrain. Two days after fertilization (stage 10), the clusters of positive neurons showed labeled axons. The two longitudinal fiber bundles that arose from the diencephalic positive neurons ran caudally in the tract of the postoptic commissure. At stage 12 (3.5 days after fertilization), new populations of NADPH-diaphorase-positive neurons appeared in the telencephalon, in some diencephalic nuclei, and in the hypothalamus. Several trigeminal motor neurons showed strong NADPH-diaphorase activity, whereas the optic tectum and cerebellum were completely free of enzymatic activity. In the hindbrain, clusters of positive neurons were seen in the octavolateral region and in the region defined by the exit of the vagus nerve. In the cervical spinal cord, some ventral putative motor neurons were labeled. At stage 14 (5.5 days after fertilization), several periventricular neurons of the optic tectum and some neurons of the cerebellar lamina were labeled. Dorsal neurons, including a few large superficial neurons were also labeled in the cervical spinal cord. NADPH-diaphorase activity was seen in the neuropil area of the telencephalon, the target of olfactory inputs, and in the sensory dorso-lateral area of the spinal cord.     

Species specificity in the responsiveness of chick embryo neural tube explants to target-conditioned medium

Explants from the metencephalic region of 40-h chick embryo neural tubes containing the trigeminal (V) motor nucleus were cultured in appropriate target muscle-conditioned media (MCM) derived from chick, quail and rat embryos. Enhanced neuritic outgrowth was found only in the presence of chick MCM, indicating that this early, initial responsiveness to target-released materials within this system is species-specific.     

The Nrf2-inducible antioxidant defense in astrocytes can be both up- and down-regulated by activated microglia:Involvement of p38 MAPK

The effects of microglia-conditioned medium (MCM) on the inducible Nrf2 system in astrocyte-rich cultures were investigated by determination of glutathione (GSH) levels, γglutamylcysteine ligase (γGCL) activity, the protein levels of Nrf2, Keap1, the modulatory subunit of γGCL (γGCL-M) and activated MAP kinases (ERK1/2, JNK and p38). Microglia were either cultured for 24 h in serum-free culture medium to achieve microglia-conditioned medium from non-activated cells (MCM(0) ), used as control condition, or activated with different concentrations (0.1-1,000 ng mL(-1) ) of lipopolysaccharide (LPS) to produce MCM(0.1-1,000) . Acute exposure (24 h) to MCM(100) increased GSH, γGCL activity, the protein levels of γGCL-M, Nrf2, and activated JNK and ERK1/2 in astrocyte-rich cultures. In contrast, treatment with MCM(10) for 24 h decreased components of the Nrf2 system in parallel with activation of p38 MAPK. Stimulation of the Nrf2 system by tBHQ was partly intact after 24 h but blocked after 72 h treatment with MCM(10) and MCM(100) . This down-regulation after 72 h correlated with activation of p38 MAPK and lack of ERK1/2 and JNK activation. The negative effects were partly reversed by an inhibitor of p38 which restored tBHQ mediated protection against oxidative stress. In conclusion, the study showed a negative effect of MCM(10) on the inducible anti-oxidant defense in astrocyte-rich cultures at both 24 and 72 h that correlated with activation of p38 and was partly reversed by a p38 inhibitor. A transient protective effect of MCM(100) on astrocyte-rich cultures against H(2)O(2) toxicity was observed at 24 h which coincided with activation of JNK and ERK1/2.     

Jaw tremor: prevalence and clinical correlates in three essential tremor case samples.

The spectrum of involuntary movements seen in essential tremor (ET) is limited. Jaw tremor is one such movement. The prevalence and clinical correlates of jaw tremor have not been studied in detail. The objective of this study was to estimate the prevalence and examine the clinical correlates of jaw tremor in ET using ET cases from three distinct settings (population, tertiary-referral center, brain repository). All ET cases underwent a videotaped tremor examination in which tremors (including limb, head, voice, and jaw) were assessed. The prevalence [95% confidence interval (CI)] of jaw tremor was lowest in the population sample (7.5%; 3.9%-14.2%), intermediate in the tertiary-referral center (10.1%; 6.8%-14.7%), and highest in the brain repository (18.0%; 12.3%-25.5%; P = 0.03). Jaw tremor was associated with older age (P < 0.001), more severe action tremor of the arms (P < 0.001), and presence of head and voice tremor (P < 0.001). Jaw tremor was present in 4/14 (28.6%) ET cases with consistent rest tremor vs. 15/193 (7.8%) cases without rest tremor (odds ratio = 4.8; 95% CI = 1.3-7.0; P = 0.009). The prevalence of jaw tremor was 7.5% to 18.0% and was dependent on the mode of ascertainment, being least prevalent in a population-based sample. ET cases with jaw tremor had a more clinically severe and more topographically widespread disorder. The association in our study between jaw tremor and rest tremor, along with the published observation that jaw tremor can occur in Parkinson's disease (PD), raises the question whether jaw tremor in ET is a marker for subsequent conversion to PD.     

Developmental regulation of chick embryo retina neurite extension by extrinsic factors

Developmental changes in the control of neurite extension by extracellular factors can be examined by utilizing cultures of neurons from various aged embryos. Several conditioned media and tissue extracts were added to cultures of chick embryo retinal neurons on collagen substrates for 1, 3 and 5 days in vitro. Neurite outgrowth, measured as the percentage of neurons with neurites and the length of neurites, was promoted by optic tectal extract and cornea conditioned medium in retina neurons from younger ages (6- to 12-day embryos), but not from older ages (14- and 16-day embryos). The promotion of neurite outgrowth by optic tectal extracts may be mediated by a promotion of glial cell growth. The developmental changes in neurite extension may be due to either an altered sensitivity to the neurite promoting factors or by an altered intrinsic ability of retinal neurons to extend processes.     

An improved method for the expression of TRH in serum-supplemented primary cultures of fetal hypothalamic cells

Primary cultures of dispersed cells from fetal nervous tissue are extensively used for studying multiple neuronal properties. Analyses of the developmental expression of thyrotropin releasing hormone (TRH; pglu-his-pro-NH(2)) biosynthesis in primary cultures of fetal dissociated hypothalamic cells have shown that cellular TRH levels per dish increase with time in culture, after a lag period of a few days, but do not attain the values observed in vivo, hampering its use as a model system for the study of peptide biosynthesis and release. We have demonstrated that homologous conditioned medium (CM) enhances TRH expression in dissociated cell cultures from fetal mice hypothalamus, maintained in presence of serum. We report here experimental conditions that allow the expression, during the second or third week in vitro, of higher cellular TRH levels than previously described in primary cultures of dissociated hypothalamic cells from 17-day rat fetuses. The medium used was Dulbecco's Modified Eagle Medium (DMEM) supplemented with fetal bovine serum (10%), vitamins, glucose, glutamine and insulin (DMEM-S). Cellular levels of TRH/mg protein increased with cell density between 1 and 2.7x10(6) cells per 35-mm dish. Addition of 10(-5) M cytosine arabinoside (CAr) at the 4th day in vitro (DIV) improved TRH cell content per dish compared to addition at 5 DIV; 2.5-5x10(-5) M bromodeoxyuridine added at seeding reduced cell survival and did not enhance TRH levels, in comparison to CAr-treated cultures. Addition of ascorbic acid (0.5-1x10(-4) M) increased TRH levels per dish. Substitution of DMEM by DMEM-F12 (1:1) did not improve TRH levels. Cellular levels of TRH, in Neurobasal plus B27 (a serum-free medium), were similar to levels in serum-supplemented media. In the optimized conditions, a small number of pro-TRH mRNA expressing cells (2% of total cells) was detected by in situ hybridization; 40% coexpressed the pro-protein convertase PC1 mRNA. Conditioning the medium, controlling glial proliferation, and adding ascorbic acid improved the expression of TRH in primary culture of hypothalamic cells in DMEM-S.     

Perinatal asphyxia induces neurogenesis in hippocampus: an organotypic culture study

There is clinical and experimental evidence indicating that neurocircuitries of the hippocampus are vulnerable to hypoxia/ischemia occurring at birth, inducing, upon re-oxygenation/re-circulation, delayed neuronal death, but also compensatory mechanisms, including neurogenesis. In the present report, perinatal asphyxia was induced by immersing foetuses-containing uterine horns removed from ready-to-deliver rats into a water bath at 37 degrees C for 20 min. Some pups were delivered immediately after the hysterectomy to be used as non-asphyxiated caesarean-delivered controls. The pups were sacrificed after seven days for preparing organotypic hippocampal cultures. The cultures were grown on a coverslip in a medium-containing culture tube inserted in a hole of a roller device standing on the internal area of a cell incubator at 35 degrees C, 10% CO2. At days in vitro (DIV) 25-27, cultures were fixed for assaying cell proliferation and neuronal phenotype with antibodies against 5-bromo-2'deoxyuridine (BrdU) and microtubule associated protein-2 (MAP-2), respectively. Confocal microscopy revealed that there was a 2-fold increase of BrdU-positive, but a 40% decrease of MAP-2-positive cells/mm3 in cultures from asphyxia-exposed, compared to that from control animals. Approximately 30% of BrdU-positive cells were also positive for MAP-2 (approximately 4800 cells), mainly seen in the dentate gyrus of the hippocampus, demonstrating a 3-fold increase of postnatal neurogenesis, when the total amount of double-labelled cells seen in cultures from asphyxia-exposed animals is compared to that from control animals.     

Differential effects of one and repeated endotoxin treatment on pituitary- adrenocortical hormones in the mouse: role of interleukin-1 and tumor necrosis factor-alpha.

The role of endogenous interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha) in modulating the hypothalamic-pituitary-adrenal (HPA) axis response was examined in male C57BL/6 mice injected with endotoxin (lipopolysaccharide, LPS) or saline at 24-hour intervals for 4 or 8 consecutive days. The mice were divided into four groups: (1) LPS injections for 4 or 8 days and LPS injection on day 5 or 9, respectively (LPS-LPS); (2) LPS injections for 4 or 8 days and saline injection on day 5 or 9, respectively (LPS-saline); (3) saline injections for 4 or 8 days and LPS injection on day 5 or 9, respectively (saline-LPS), and (4) saline injections for 4 or 8 days and saline injection on day 5 or 9 (saline-saline). The mice were sacrificed by decapitation 2 h after the last injection and plasma levels of hormones and cytokines and tissue levels of IL-1beta were measured. Plasma adrenocorticotropin (ACTH) levels were significantly attenuated in the LPS-LPS group compared with the dramatic increases in the saline-LPS group following 4 or 8 days of endotoxin treatment. Plasma corticosterone concentrations were comparable in the LPS-LPS group after 4 days' treatment, but significantly lower following 8 days of treatment when compared with saline-LPS group. Repeated endotoxin treatment followed by a single saline injection (LPS-saline) did not alter the levels of IL-1beta in plasma or any of the tissues examined. IL-1beta levels in the hippocampus, hypothalamus, adrenal gland and plasma were elevated to comparable levels in the saline-LPS and LPS-LPS groups after 4 days of treatment. In contrast to the plasma IL-1beta response, TNFalpha levels were dramatically increased in the saline-LPS group but not in the LPS-LPS group following the 4-day treatment regimen. Increases in IL-1beta concentrations were seen in all tissues following one endotoxin challenge in the saline-LPS group following the 8-day treatment regimen, while increases were significantly attenuated in the hypothalamus, adrenal gland and plasma in LPS-LPS for 8 days. The sustained increases in tissue levels of IL-1beta following 4-day endotoxin treatment appears to have functional consequences since [125I]IL-1alpha binding was significantly decreased in the LPS-saline group compared with the saline-saline group. Furthermore, [125I]IL-1alpha binding was markedly reduced in the LPS-LPS group compared with the saline-LPS group. There was a significant positive correlation between plasma ACTH and IL-1beta after a single and repeated LPS treatment for 4 days, while a significant correlation was seen between plasma ACTH and TNFalpha following one but not repeated LPS treatment. These data demonstrate a differential regulation of IL-1beta and TNFalpha by repeated endotoxin treatment and suggest that while TNFalpha may be important modulating the attenuated pituitary adrenocortical response following the 4-day endotoxin treatment, IL-1beta appears to be the primary regulator of the response following the 8-day endotoxin treatment in the regulation of the HPA axis.     

小鼠卵巢器官不同部位移植的效果比较

背景：最佳移植部位的选择对于保证卵巢的存活起着很重要的作用。 目的：比较小鼠卵巢器官不同移植部位自体移植后的生长发育情况。 设计、时间及地点：动物实验观察，于2008-09/11在宁夏医科大学生殖与遗传重点实验室完成。 材料：清洁级健康ICR品系雌性小白鼠60只，按照不同的移植部位随机分为对照组、自体肾被膜下、颈部皮下、腹腔内及下肢根部肌肉内移植组，12只/组。 方法：①对照组：新鲜取卵巢后立刻固定。各移植组在切除双侧卵巢后，立即进行自体异位移植。②肾被膜移植组：在肋脊角处逐层切开两侧的皮肤和肌肉，充分暴露小鼠双侧肾脏，用显微手术镊将完整卵巢由肾被膜切口移入肾脏下端。③颈部皮下移植组：在颈部双侧靠近颈动脉处切口，钝性分离皮下组织，用缝线将附近结缔组织缝成荷包，将完整卵巢植入荷包内并缝合。④腹腔移植组：将卵巢移植于腹腔子宫角系膜处。⑤下肢根部肌肉组织移植组：移植前4 d分别在小鼠左右下肢根部作一切口，用小弯钳在肌层内进行钝性分离制备一创面后，缝合切口。4 d后进行自体卵巢移植时，将卵巢组织移进肌层预制的创面内，缝合切口。 主要观察指标：于移植后48，168 h取移植卵巢进行组织学和血供重建分析。 结果：移植48 h后，肾被膜下移植组卵巢与移植部位重建血供，而下肢根部肌肉移植组无血供建立；移植168 h，腹腔移植组的卵泡计数最低，与对照组相比有显著性差异(P < 0.05)；下肢根部肌肉移植组卵泡结构正常，但数目较肾被膜移植组及颈部移植组少，但差异无显著性(P > 0.05)。肾被膜下移植组和颈部皮下移植组存活的卵巢器官内均可见不同发育阶段的卵泡，形态正常，与对照组相近。 结论：卵巢组织的肾被膜下及颈部皮下移植较腹腔及下肢肌肉移植更易重建血供并利于卵泡的发育。     

Gangliosides of the mouse spinal cord: a comparison in in vivo and in vitro tissues

Ganglioside profiles in spinal cord from 13-day mouse fetuses, 21-day postnatal and adult mice were compared with those harvested from organotypic cross-sections of fetal mouse spinal cord grown for 28 days in vitro in a serum-free medium. All the major species of gangliosides reported for brain were present both in the in vivo tissue and cultured spinal cord, though not necessarily at each developmental stage examined. Fresh tissues showed increases and decreases in various gangliosides as have been reported for higher brain centers at similar stages of development in mammals and birds. However, qualitative and quantitative differences exist between fresh spinal cord and cultured cord explants as well as between galactose-grown and galactose-free cultures. Spinal cord explants grown in the presence of galactose showed measurable amounts of GM2 and GM3 which were not detected in the control-defined medium-grown cultures. The differences between the two culture groups may be related to interneuronal connectivity patterns.     

The lazaroid U-83836E improves the survival of rat embryonic mesencephalic tissue stored at 4°C and subsequently used for cultures or intracerebral transplantation

We assessed the effects of addition of the lazaroid U-83836E to a preservation medium on the survival of rat dopamine neurons stored before culturing or intracerebral transplantation. Embryonic ventral mesencephalic tissue was preserved at 4°C for 8 days with or without the addition of 0.3 μM of U-83836E to a chemically defined “hibernation” medium. Freshly dissected mesencephalic tissue was used in control groups. For culture experiments, the mesencephalic tissue was dissociated and grown in serum-containing medium. Following 24–48 h in vitro, the number of dopamine neurons in cultures derived from tissue hibernated without the lazaroid was 40% of fresh control, compared with 67% of control in cultures prepared from tissue stored in the presence of U-83836E. When mesencephalic tissue was transplanted to the dopamine-depleted striatum of hemiparkinsonian rats following 8 days storage at 4°C in a medium without U-83836E, the mean number of surviving dopamine neurons in the grafts was significantly reduced to 40% of control. In contrast, grafts of tissue which had been hibernated in U-83836E-containing medium contained as many dopamine neurons as transplants of freshly dissected tissue. High yields of surviving grafted dopamine neurons were correlated to a significantly faster onset of functional recovery of amphetamine-induced motor asymmetry. We conclude that the storage period for rat mesencephalic tissue can be prolonged up to 8 days when using lazaroid-supplemented hibernation medium. As lazaroids have undergone clinical safety testing, the application of lazaroids for tissue storage in clinical transplantation trials can be envisaged.