Implantation of embryonic brain tissue into regenerating peripheral nerve

Dynamics of development of the cerebral cortex tissue anlage in 17-day-old embryos of Wistar rats, implanted into the sciatic nerve of mature rats with the aim to establish new relay and trophic centers in the regenerating nerve have been studied. By means of certain morphological methods (silver nitrate impregnation after Bielschowsky-Gros, Sudan black, hematoxylin-eosin, toluidine blue after Nissl stainings) it has been stated that the implanted nerve cells not only preserve their viability, but also differentiate from neuroblasts up to young and mature neurons during 2 months. Already in 14 days after the operation there are blood vessels in the implants; by the 2d month massive myelinization of axons begins in the implant. A part of the regenerating myelin fibers of the nerve gets into the implant and branches. In similar cases connections between the implanted neurons and the host peripheral nervous fibers are supposed to be established.     

Development of the fragments of embryonal spinal cord in a damaged peripheral nerve of a mature animal

By means of morphological methods dynamics of the spinal cord development in 14-day-old rat embryos implanted into the sciatic nerve of mature rats have been studied. The implants preserve their viability during 5 months after the operation and their cells continue to differentiate beginning from neuroepithelial cells and neuroblasts up to young and mature neurons with histotypical signs of motoneurons. In 6 h and 1 day after transplantation the neuroepithelial cells continue their mitotic division. In 3 days, however, their mitotic activity decreases essentially and differentiation of neuroblasts begins. In 7 days the implants consist mainly of differentiated neuroblasts and glial cells. As demonstrates electron microscopy, in 30 days after the operation in the implants there is a well developed neuropil, where mature neurons, myelinated axons are situated and synaptic contacts are present.     

Permeability of the normal rat brain,spinal cord and dorsal root ganglia microcirculations to immunoglobulins G

The distribution of blood-borne immunoglobulins G (IgG) was studied in the cerebral cortex, pineal gland, spinal cord and dorsal root ganglia of normal Lewis rats using the detection of autologous anti-horseradish peroxidase (HRP) antibodies. This detection was performed by means of light and electron microscopy. This study demonstrated that, in the cerebral cortex and the spinal cord microcirculations, endothelial cells are a restrictive barrier against IgG while IgG are able to diffuse into the perivascular parenchyma of the pineal gland and spinal ganglia.     

Permeability of the normal rat brain, spinal cord and dorsal root ganglia microcirculations to immunoglobulins G

The distribution of blood-borne immunoglobulins G (IgG) was studied in the cerebral cortex, pineal gland, spinal cord and dorsal root ganglia of normal Lewis rats using the detection of autologous anti-horseradish peroxidase (HRP) antibodies. This detection was performed by means of light and electron microscopy. This study demonstrated that, in the cerebral cortex and the spinal cord microcirculations, endothelial cells are a restrictive barrier against IgG while IgG are able to diffuse into the perivascular parenchyma of the pineal gland and spinal ganglia.     

CHANGES IN CENTRAL NORADRENALINE NEURONSADMINISTRATION AFTER SYSTEMIC 6-HYDROXYDOPAMINE ADMINISTRATION

—Intravenous injection of a large dose of 6-hydroxydopamine (100 mg/kg) to adult rats caused a significant and long-lasting reduction (about 30 per cent) of the in oirro uptake of [³H]NA in the cerebral cortex and spinal cord, while no changes were seen in the hypothalamus. The endogenous NA in whole brain was similarly reduced (about 20 per cent). Fluorescence histochemistry revealed catecholamine accumulations which are degenerative signs, induced by 6-hydroxydopamine, in axons of the dorsal NA bundle innervating the cerebral cortex. It is concluded that the blood–brain barrier in adult rats is not completely protective with respect to the neurotoxic action of systemically injected 6-hydroxydopamine, which can produce degeneration of a significant number of NA nerve terminals in the cerebral cortex and spinal cord. Previous studies have shown that 6-hydroxydopamine caused a permanent and selective degeneration of a large number of central NA nerve terminals when injected systemically up to 1 week after birth, due to an incompletely developed blood-brain barrier. This barrier for 6-hydroxydopamine develops between the 7th and 9th day after birth (Sachs , 1973). In the present study 6-hydroxydopamine was found to cause a small transient reduction in [³H]NA uptake in cerebral cortex of rats between 9 and 28 days of age, while in older rats the damage produced by 6-hydroxydopamine was long-lasting. Thus, the NA nerves ascending to the cerebral cortex seem to possess a regenerative capacity to a 6-hydroxydopamine-induced degeneration up to about 28 days postnatally, but which later disappears or is markedly retarded.     

Vitamin E concentrations in different regions of the spinal cord and sciatic nerve of the rat

Since the spinal cord and peripheral nerves are vulnerable to the effects of vitamin E deficiency, vitamin E concentrations in various discrete regions of these parts of the nervous system of the rat were determined. Furthermore, as acrylamide toxicity and vitamin E deficiency share some neuropathological features, tissue vitamin E concentrations in acrylamide-treated rats were also studied. Male Sprague Dawley rats (200 to 250 g body weight) were fed normal rat chow with or without 0.03% acrylamide in their drinking water. After 24 days, the animals were sacrificed and the tissues assayed for vitamin E by a liquid chromatographic method. Vitamin E concentrations decreased from cerebral cortex to spinal cord with no concentration gradients between different levels of the spinal cord. Sciatic nerve concentration of alpha tocopherol was as high as that of cerebral cortex, and the former also contained measurable amounts of gamma tocopherol. Vitamin E concentrations in the majority of nervous tissue samples remained unchanged with acrylamide treatment.Presented in part at the Sixteenth Annual Meeting of the American Society for Neurochemistry, March 1985.     

Afferent and efferent connections of cortical transplants implanted into the damaged sensorimotor area of the cerebral cortex of mature rats

Afferent and efferent connections of the transplant, implanted in the previously damaged sensorimotor area of the mature rat cerebral cortex have been studied by means of axonal transport of horseradish peroxidase. For 5 months after transplantation neural axons of the transplant are capable to reach the caudo-putamen and thalamic structures, while connections with the spinal cord are absent. The afferent connections of the transplant are minimal and belong only to the neighbouring areas of the cortex and the caudo-putamen of the recipient brain. Presence of efferent projections to the striate and thalamic structures demonstrates specificity of the projections formed; this can be a morphological base for restoration of the functions lost after the damage of the sensorimotor area of the cortex in mature animals.     

Nerve growth factor-induced stimulation of dorsal root ganglion/spinal cord co-grafts in oculo: enhanced survival and growth of CGRP-immunoreactive sensory neurons

Intraocular co-grafts of rat fetal spinal cord and dorsal root ganglia were used to examine the enhanced survival, growth, and differentiation of sensory neurons by nerve growth factor. E14 lumbar spinal segments were implanted into the anterior eye chamber of capsaicin-pretreated rats. Two weeks later, an E14 dorsal root ganglion was implanted beside the spinal cord graft. Nerve growth factor or vehicle was injected weekly for 4 weeks into the anterior eye chamber. Co-grafts were examined weekly and, at 6 weeks, processed for calcitonin gene-related peptide (CGRP) immunofluorescence. No differences in overall size were determined for the grafts. Co-grafts treated with nerve growth factor contained many more CGRP neurons (19.4 cells/20 microm) that were significantly larger (mean 764 microm2) than neurons from control co-grafts (8.6 cells/20 microm; mean 373 microm2). In co-grafts treated with nerve growth factor, CGRP-immunoreactive fibers were extensive in the dorsal root ganglion, adjacent iris, and spinal cord compared to control co-grafts. A few CGRP-positive motoneurons were observed in the spinal cord, but no differences in number or size of motoneurons were found. The current report demonstrates that spinal cord and dorsal root ganglia can be co-grafted in oculo for long periods of time. Many dorsal root ganglion neurons survive and send peripheral processes into the iris and central processes into the spinal cord under the influence of exogenous nerve growth factor. The intraocular graft paradigm can be of use to further examine the role of neurotrophic factors in regulating or modulating dorsal root ganglion and spinal cord neurons.     

神经移植的新途径--移植的中枢神经元从蛛网膜下腔迁入脊髓和大脑皮层

我们在神经移植的天空过程中观察到被移植的中枢神经元能从蛛网膜下腔迁入脊髓的大脑皮层。这一新观察为脊髓和脑浅层大范围神经元缺损时的无损伤神经元引入和大范围去神经区域的神经再支配提供了一种颇具吸引力的河能性。实验动物选用Ｗｉｓｔａｒ和Ｓ．Ｄ．大鼠,将含有胚胎中枢单胺或精氨酸血管加压素（ＡＶＰ）能神经元的细胞悬浮液或组织块移植到被横断的脊髓或未被脊髓和脑的蛛网膜下腔内。动物分别在移植的同时切断脊髓;在移     

Distribution of FMRFamide-like immunoreactivity in the central nervous system of the Formosan monkey (Macaca cyclopsis)

The distribution of FMRFamide-like immunoreactivity in the central nervous system of the Formosan monkey (Macaca cyclopsis) was investigated employing immunohistochemical techniques. FMRFamide-containing cells were found to be widely distributed throughout the forebrain. Principal densities of FMRFamide neuronal perikarya were observed in the following areas: the amygdaloid complex, the olfactory tubercle, the cerebral cortex, the basal ganglia, the septum, the caudate-putamen and the arcuate nucleus. A large number of immunoreactive fibers were observed in areas ranging from the cerebral cortex to the spinal cord, and were noted in the following locations: the preoptic area, the tuberal and posterior hypothalamic areas, the bed nucleus of the stria terminalis, the nuclei of the spinal trigeminal nerve, the hypoglossal nucleus, the nucleus of the solitary tract, and the dorsal horn of the spinal cord. The results generally parallel those described in the rat and guinea pig.     

Effect of neonatal 6-hydroxydopamine treatment on audiogenic seizures

Subcutaneous injection of 6-hydroxydopamine (6-OHDA) in newborn audiogenic rats resulted in an increase in convulsive seizure intensity and a decrease in norepinephrine concentration in the cerebral cortex and the spinal cord. In addition, norepinephrine concentration in the brainstem (pons-medulla) was increased. Dopamine concentration in all brain regions studied was unchanged. The results suggest that norepinephrine exerts its modulatory influence on convulsive seizures by an action in either the spinal cord, the cerebral cortex, or both.     

胚胎神经干细胞移植及胶质细胞源性神经营养因子对大鼠脊髓损伤的修复作用

将胚胎神经干细胞(neural stem cells,NSCs)移植至成年大鼠损伤的脊髓,观察移植后NSCs的存活、迁移以及损伤后的功能恢复。实验结果显示：动物NSCs移植4周后,斜板实验平均角度和运动评分结果比对照组均有明显增高(P＜0．05),而脊髓损伤(spinal cord injury,SCI)处的空洞面积显著减小(P＜0．05);在NSCs中加入胶质细胞源性的神经营养因子(glial cell line-derived neurotrophic factor,GDNF)后,上述改变更加显著。移植后的NSCs不仅能存活,而且向损伤的头端和尾端迁移达3mm之远。这些结果表明,移植的NSCs不仅可以存活、迁移,还可减小SCI空洞面积,促进动物神经功能的恢复;此外,我们的结果还表明GDNF对SCI功能恢复有促进作用。     

Evaluation of neuroactive steroid levels by liquid chromatography-tandem mass spectrometry in central and peripheral nervous system: effect of diabetes

The nervous system is a target for physiological and protective effects of neuroactive steroids. Consequently, the assessment of their levels in nervous structures under physiological and pathological conditions is a top priority. To this aim, identification and quantification of pregnenolone (PREG), progesterone (PROG), dihydroprogesterone (DHP), tetrahydroprogesterone (THP), testosterone (T), dihydrotestosterone (DHT), 5alpha-androstan-3alpha, 17beta-diol (3alpha-diol), 17alpha- and 17beta-estradiol (17alpha-E and 17beta-E) by liquid chromatography and tandem mass spectrometry (LC-MS/MS) has been set up. After validation, this method was applied to determine the levels of neuroactive steroids in central (i.e., cerebral cortex, cerebellum and spinal cord) and peripheral (i.e., brachial nerve) nervous system of control and diabetic rats. In controls only the brachial nerve had detectable levels of all these neuroactive steroids. In contrast, 17alpha-E in cerebellum, 17alpha-E, 17beta-E, DHP and THP in cerebral cortex, and 17alpha-E, 17beta-E and DHP in spinal cord were under the detection limit. Diabetes, induced by injection with streptozotocin, strongly affected the levels of some neuroactive steroids. In particular, the levels of PREG, PROG and T in cerebellum, of PROG, T and 3alpha-diol in cerebral cortex, of PROG, DHT and 3alpha-diol in spinal cord and of PREG, DHP, THP, T, DHT and 3alpha-diol in brachial nerve were significantly decreased. In conclusion, the data here reported demonstrate that the LC-MS/MS method allows the assessment of neuroactive steroids in the nervous system with high sensitivity and specificity and that diabetes strongly affects their levels, providing a further basis for new therapeutic tools based on neuroactive steroids aimed at counteracting diabetic neuropathy.     

Developmental regulation of GABA(B) receptor function in rat spinal cord

GABA(B) receptors are heterodimers coupled to G-proteins. The present study was undertaken to investigate activation of GABA(B) receptors in cerebral cortex and spinal cord using [35S]GTPgammaS binding assays, a direct measure of G-protein activity. The results revealed that the GABA(B) agonist baclofen stimulates GTPgammaS binding in cerebral cortex, with an ED50 of 50microM. This response is blocked by the GABA(B) receptor antagonist CGP 55845A (100nM). In contrast, baclofen-stimulated GTPgammaS binding was not observed in adult spinal cord tissue under similar incubation conditions, or after varying magnesium, calcium, GDP, [35S]GTPgammaS, or membrane concentrations in the assay medium. Stimulation of adult rat spinal cord muscarinic receptors did result in a concentration-related increase in [35S]GTPgammaS binding. Baclofen-stimulated GTPgammaS binding in adult spinal cord did not appear after peripheral inflammation, despite significant increases in GABA(B) subunit mRNA levels. As opposed to adult, appreciable GTPgammaS binding was observed in membranes prepared from spinal cords of rats within the first 14 days of postnatal development, suggesting that GABA(B) receptor function in the rat spinal cord is developmentally regulated. The results indicate that GABA(B) receptors may not be coupled to G-proteins in the adult rat spinal cord, or couple in a way that differs from that in newborns or adult cerebral cortex.     

Distribution of stomatin expressing in the central nervous system and its up-regulation in cerebral cortex of rat by hypoxia

Stomatin is an important membrane raft protein which can combine skeleton protein, some ion channel, and transporter to regulate their functions. However, until now no data on its expression and function in CNS are available. In this study, we examined distribution of stomatin in CNS of rat, and investigated the effects of hypoxia exposure and glucocorticoid on stomatin expression in cerebral cortex of rat. Immunofluorescence staining revealed a broad expression of stomatin protein in many areas of adult rat brain and spinal cord, including the ventral horn of spinal cord, causal magnocellular nucleus of hypothalamus, the V layer of the cerebral cortex, solitary nucleus, 10 and 12 nuclei, and so on. Hypoxia or ischemic hypoxia significantly up-regulated stomatin expression in cerebral cortex, and the up-regulation was independent on adrenocortical steroids since it also occurred in adrenalectomized (ADX) rats. Moreover, treatment of ADX or sham-operated rats with dexamethasone, a synthetic glucocorticoid alone could significantly stimulate expression of stomatin in lung and heart, but not in cerebral cortex. However, dexamethasone could enhance the hypoxia-stimulated expression of stomatin in cerebral cortex of ADX rats. These findings suggested that stomatin might be involved in various physiological functions and cellular events of neurons in CNS under physiological conditions and play a potential protective role under hypoxic conditions.     

Is Remodelling of Corticospinal Tract Terminations Originating in the Intact Hemisphere Associated with Recovery following Transient Ischaemic Stroke in the Rat?

Following large strokes that encompass the cerebral cortex, it has been suggested that the corticospinal tract originating from the non-ischaemic hemisphere reorganises its pattern of terminal arborisation within the spinal cord to compensate for loss of function. However many strokes in humans predominantly affect subcortical structures with minimal involvement of the cerebral cortex. The aim of the present study was to determine whether remodelling of corticospinal terminals arising from the non-ischaemic hemisphere was associated with spontaneous recovery in rats with subcortical infarcts. Rats were subjected to transient middle cerebral artery occlusion or sham surgery and 28 days later, when animals exhibited functional recovery, cholera toxin b subunit was injected into the contralesional, intact forelimb motor cortex in order to anterogradely label terminals within cervical spinal cord segments. Infarcts were limited to subcortical structures and resulted in partial loss of corticospinal tract axons from the ischaemic hemisphere. Quantitative analysis revealed there was no significant difference in the numbers of terminals on the contralesional side of the spinal grey matter between ischaemic and sham rats. The results indicate that significant remodelling of the corticospinal tract from the non-ischaemic hemisphere is not associated with functional recovery in animals with subcortical infarcts.     

Expression of Bcl-2, Bax and Caspase-3 in Nerve Tissues of Rats Chronically Exposed to 2,5-hexanedione

Occupational exposure and experimental intoxication with n-hexane or its metabolite 2,5-hexanedione (HD) produce a central-peripheral neuropathy. However, the mechanism remains unknown. We hypothesized that HD affected the expression of Bcl-2, Bax and Caspase-3 in the central nervous system (CNS) and the peripheral nervous system (PNS). Male adult Wistar rats were administered by intraperitoneal injection at a dosage of 200 or 400 mg/kg HD, five days per week for 8 weeks. Samples of the cerebral cortex, cerebellum, spinal cord and sciatic nerves were collected and examined for Bcl-2, Bax and Caspase-3 expression using Western blotting. Subchronic exposure to HD resulted in significantly increased expression of both anti-apoptotic protein Bcl-2 and pro-apoptotic protein Bax and Caspase-3 in cerebral cortex and cerebellum, which exhibited a dose-dependent pattern. Though little change was detected in spinal cord, our results showed that the expression of Bcl-2, Bax and Caspase-3 was markedly enhanced in the sciatic nerves. These findings suggested that the changes of apoptosis-related protein level in rat nerve tissues were associated with the intoxication of HD, which might be involved in early molecular regulatory mechanism of apoptosis in the HD-induced neuropathy.     

移植的5—羟色胺能神经元跨软脊膜迁入大鼠脊髓的研究

本文对移植的5-HT神经元从蛛网膜下腔跨软脊膜迁移进入脊髓作了初步研究。将含有5-HT细胞的胚胎中缝核组织小块或神经细胞悬浮液作为移植物,以5-HT免疫组织化学方法跟踪移植细胞,结果如下:(1)在低胸水平横切脊髓,10d后,横断脊髓内的5-HT纤维消失。(2)横切脊髓(方法同上)后,立即将中缝核组织小块移植在胸腰段脊髓的蛛网膜下腔,一月后.在横断脊髓内出现5-HT阳性神经元和纤维。5-HT纤维能在灰白质内延伸。(3)脊髓横断后,若以中缝核的细胞悬浮液代替组织小块,作上述移植,则在移植区附近的灰质内出现大量的5-HT阳性神经元。这些神经元在灰质内的分布范围与神经细胞悬浮液在蛛网膜下腔的移植范围相一致。迁入神经元能在灰质内重新形成5-HT阳性纤维网。(4)经上述移植后,灰质内出现的5-HT阳性纤维随远离细胞体而变得稀疏。白质内的5-HT阳性纤维远比灰质内稀少。本实验结果表明:移植在脊髓蛛网膜下腔的脑干5-HT细胞能跨软脊膜迁移进入脊髓。     

Cell fate control in the developing central nervous system

The principal neural cell types forming the mature central nervous system (CNS) are now understood to be diverse. This cellular subtype diversity originates to a large extent from the specification of the earlier proliferating progenitor populations during development. Here, we review the processes governing the differentiation of a common neuroepithelial cell progenitor pool into mature neurons, astrocytes, oligodendrocytes, ependymal cells and adult stem cells. We focus on studies performed in mice and involving two distinct CNS structures: the spinal cord and the cerebral cortex. Understanding the origin, specification and developmental regulators of neural cells will ultimately impact comprehension and treatments of neurological disorders and diseases.     

DEVELOPMENT OF THE BLOOD-BRAIN BARRIER FOR 6-HYDROXYDOPAMINE

The postnatal development of the blood-brain barrier for the neurotoxic action of 6-hydroxydopamine on central noradrenaline neurons has been investigated by recording the in vitro uptake of [³H]noradrenaline in slices from cerebral cortex, hypothalamus and spinal cord in rats treated with large doses of 6-hydroxydopamine at different ages. The [³H]noradranaline uptake was permanently and markedly reduced in all regions when the animals were treated at birth, certainly related to degeneration of noradrenaline neurons, caused by 6-OH-DA. In the cerebral cortex and hypothalamus an efficient protection against the effects of 6-OH-DA on [³H]noradrenaline uptake developed postnatally, while in the spinal cord this protection was never seen to become complete. The results obtained indicate a rapid formation of a blood-brain barrier for 6-OH-DA in the cerebral cortex between the 7th and 9th day after birth. In the hypothalamus the development of this barrier seemed to have a more gradual time-course, but appeared to be fully developed already at day 5 postnatally. Also in the spinal cord the barrier developed more gradually from birth to the adult age. It was observed, however, that both in the cerebral cortex and in the spinal cord, the blood-brain barrier developed, could not completely protect the central noradrenaline neurons from the neurotoxic actions of large doses of 6-OH-DA administered systemically to adult rats. Furthermore, the results obtained support the view that 6-OH-DA does not seem to apparently affect the outgrowth of remaining NA neurons which have not been destroyed by the 6-OH-DA treatment.