Surface structure of nerve tissue transplants growing in the anterior chamber of the rat eye (electron-microscopic study)

Two different types of the glial envelope are demonstrated around the septal cerebral area grafts (SCAG) and the hippocampal grafts (HG) developing in the anterior eye chamber of the rat during 3-4 months. In the SCAG it is multilayered, consists mainly of modified oligodendrocytes. Processes of the nervous cells perforate the surface of the envelope. In the HG the unilayered glial envelope consists of bodies and processes of the fibrous astrocytes; it is impenetrable for the nervous processes. Since both types of the cellular elements making these envelopes have been described in the norm, it is possible that the differences in the surfaces described are specific for these structures.     

Intra-Retrosplenial Cortical Grafts of Cholinergic Neurons: Functional Incorporation and Restoration of High Affinity Choline Uptake

Fetal septal neurons transplanted into the deafferented retrosplenial cortex (RSC) of rats have been shown to reinnervate the host brain and ameliorate spatial memory deficits. In the present study we examined the effects of implanting cholinergic neurons on high affinity choline uptake (HACU) in the denervated RSC and the correlational relationship between this cholinergic parameter and the level of behavioral recovery. Three groups of animals were used: 1) normal control rats (NC), 2) rats with lesions of the fornix and cingulate pathways (FX), and 3) lesioned rats with fetal septal grafts in the RSC (RSCsep-TPL). We found that intra-RSC septal grafts produced significant increases in HACU, and that recovery of HACU was significantly correlated with the improvements in the performance of spatial reference memory, spatial navigation, and spatial working memory tasks. We have also investigated the ability of the host brain to modulate the activity of the implanted neurons. In particular we evaluated the effect of the animals' performance in a 6-arm radial maze task on high affinity choline uptake (HACU). Animals in each of the NC, FX, and RSCsep-TPL groups were randomly assigned one of the following subgroups: 1) rats that performed the maze task before the determination of HACU (BEH), or 2) rats that did not perform the maze task before the determination of HACU (NON-BEH). Significant increases were observed in the NC and RSCsep-TPL groups, but not in the FX animals, indicating that fetal septal grafts in the RSC can become functionally incorporated with the host neural circuitry, and that the activity of the implanted cholinergic neurons can be modulated by the host brain.     

Nerve fiber production by intraocular adrenal medullary grafts: Stimulation by nerve growth factor or sympathetic denervation of the host iris

Summary This study evaluates the production of adrenergic nerve fibers by adrenal medullary tissue of the adult rat grafted to the anterior chamber of the eye of adult recipients. The chromaffin grafts attach to and become vascularized by the host iris. They decrease in size intraocularly during the first 3 weeks. This decrease is somewhat counteracted by sympathetic denervation of the host iris, and better counteracted by sympathetic denervation and addition of nerve growth factor (NGF, given at grafting and 1 and 2 weeks after grafting). Outgrowth of adrenergic nerve fibers from the grafts into the host iris was studied in wholemount preparations by use of the Falck-Hillarp technique 3 weeks after grafting. The innervated area of the host iris was approximately doubled in the chronically sympathectomized group and doubled again in the chronically sympathectomized NGF-supplemented group. Chronic sympathetic denervation had no effect on density of outgrowing nerves, whereas addition of NGF more than doubled nerve density. Since sympathetic denervation causes a slight elevation of NGF activity in the iris, the present experiments are taken as evidence that the level of NGF in the iris regulates formation of nerve fibers by adrenal medullary tissue grafts from adult rats.     

隔区和第Ⅲ脑室注射精氨酸加压素对家兔视前区—下丘脑前部温敏神经元放电的影响

为了研究精氨酸加压素（ＡＶＰ）的抗热机理,本研究观察了家兔隔区和第Ⅲ脑室微量注射ＡＶＰ对视前区一下丘脑前部（ＰＯ－ＡＮ）温度敏感神经元放电的影响。结果如下：（１）隔区注射ＡｖＰ能使ＰＯ－ＡＨ热敏神经元放电明显增加,冷敏神经元放电明显减少。（２）第Ⅲ脑室注射ＡＶＰ只能使部分ＰＯ－ＡＨ热敏神经元放电增加,冷敏神经元放电减少;而另外一部分热敏神经元和冷敏神经元则出现相反的效应。实验结果表明,隔区注射ＡＶＰ的抗热作用,可能是由于注射到隔区的ＡＶＰ使该区的神经元活动发生改变,而影响了ＰＯ—ＡＨ温度敏感神经元的活动所致。也提示隔区在体温调节中可能起重要作用。     

Immunochemical identification of vasopressin in neurons and in granule-containing cells of the human cerebral blood vessels

By means of indirect immunofluorescent method vasopressin localization has been defined in neurons of the locus coeruleus, in terminals, converging to the substantia nigra cells and the sylvian aqueduct area, as well as in the granule-containing cells of the human cerebral blood vessels. The granule-containing cells are identified as melanocytes, when investigated by comparative light-optic and electron microscopical technique. The melanocytes of the cerebral vessels have various size and form in accordance to their different functional state. In some melanocytes peptide vesicles are revealed. This demonstrates a possible synthesis of vasopressin. It is supposed that local endocrine regulation of the cerebral hemocirculation is performed by cooperation of granule-containing cells, producing hormones with oppositely++ directed constrictor and dilatator action to the vessels.     

Restoration of High Affinity Choline Uptake in the Hippocampal Formation Following Septal Cell Suspension Transplants in Rats with Fimbria-Fornix Lesions

High affinity choline uptake (HACU) was investigated in the hippocampal formation following fetal septal cell suspension transplants into rats with fimbria-fornix lesions. Nine-14 weeks after transplantation, HACU was markedly decreased in hippocampi from animals with fimbria-fornix lesions; this decrease was ameliorated by fetal septal cells transplanted into the host hippocampus. HACU related to septal transplantation was activated in vitro by K+, and in vivo by the administration of scopolamine and picrotoxin. These findings suggest that fetal septal cell transplantation can restore HACU in the host hippocampus following fimbria-fornix lesions, and that HACU related to the graft has pharmacological properties similar to those of the normal adult HACU system. The activation of HACU by picrotoxin, a gamma-aminobutyric acid (GABA) antagonist, suggests that transplanted cholinergic neurons receive either direct or indirect functional input from GABAergic afferents from the transplant and/or host hippocampus. Lesions of the fimbria-fornix also resulted in an increased binding to muscarinic receptors in the dorsal hippocampus. This increase in binding was not significantly ameliorated by intrahippocampal grafts of cholinergic neurons.     

Locus coeruleus fibre growth in oculo induced by trigeminotomy

Locus coeruleus from fetal donors was homologously grafted to the anterior eye chambers of adult rats whose eyes were sympathetically denervated. After intraocular maturation, outgrowth of noradrenaline-containing fibres from the locus coeruleus neurons on the host iris was studied by Falck--Hillarp fluorescence histochemistry.In control animals locus coeruleus grafts produce a halo of noradrenaline-containing nerve fibres around the graft, covering approximately one third of the surface of the host iris. Sensory denervation of host eyes carrying maturated locus coeruleus grafts was produced by intracranial lesions of the trigeminal nerve. Such lesions induced a rapid growth response in the grafted locus coeruleus neurons, leading within three weeks to complete innervation of the host iris. It was concluded that removal of non-sympathetic, non-parasympathetic nerve fibres on the host iris elicits a strong fibre-growth response in the grafted locus coeruleus.     

Immunohistochemical study on fetal raphe samples transplanted into the leptomeningeal tissue of 5,6-dihydroxytryptamine-treated adult rats

Summary Pieces of fetal midbrain raphe containing serotonergic and dopaminergic neurons were transplanted into the leptomeningeal tissue (see Fig. 3) of adult host rats that had previously been denervated by treatment with 5,6-dihydroxytryptamine. One, 2 and 5 months after transplantation, the rate of neuronal survival in the grafted tissue and the extent of axonal outgrowth into the host brain were studied by use of serotonin and tyrosine hydroxylase (TH) immunohistochemistry. The survival rate of the grafts in the 1-month group was approximately 70%. Neurons containing either serotonin or catecholamine were demonstrated by means of immunocytochemical procedures in the grafts. Two and 5 months after transplantation, serotonin-immunoreactive nerve fibers were densely distributed throughout the graft tissue, while TH-immunoreactive fiber elements were restricted to an area near the somata of TH-positive neurons. Numerous serotonin-immunoreactive fibers derived from the transplant were found in the leptomeningeal tissue surrounding the graft, on the wall of neighboring blood vessels, and also in the adjacent parenchyma of the host brain. Outgrowing TH-immunoreactive nerve fibers were not observed in the host brain, although such elements occurred in the leptomeningeal tissue and the wall of the larger blood vessels. These results suggest that the serotonergic and catecholaminergic (dopaminergic) neurons located in transplants of the raphe nuclei show different patterns when reinnervating the host tissue.     

Combined method of fluorescence tracer technique and PAP immunohistochemistry for discrimination of the transplanted cells

The retrograde fluorescence tracer, True Blue (TB), was injected into the forebrain septal area of neonatal rats. After 3 to 6 days the brains of these animals were carefully removed and placed in ice-cold sterilized physiological saline containing 1% glucose. Under the surgical microscope, one or two pairs of mesencephalic tissue samples, each containing a dorsal raphe nucleus, were punched out and transplanted into the third ventricle of a 5,6-DHT-pretreated adult rat. One month after transplantation, all animals were perfused and their brains sectioned using a cryostat. The sections were examined using a fluorescence microscope, and then processed for serotonin immunohistochemistry. The grafts were found to be successfully implanted and connected with the middle portion of the third ventricle. Four types of neurons, i.e., TB-labeled, serotonin-labeled, both TB- and serotonin-labeled, and non-labeled neurons, were detected in the grafts. This double-labeling method is considered to be a useful technique in characterizing the neurons in grafts which consist of a heterogeneous cell population.     

Reduced age-related plasticity of neurotrophin receptor expression in selected sympathetic neurons of the rat

Selective vulnerability of particular groups of neurons is a characteristic of the aging nervous system. We have studied the role of neurotrophin (NT) signalling in this phenomenon using rat sympathetic (SCG) neurons projecting to cerebral blood vessels (CV) and iris which are, respectively, vulnerable to and protected from atrophic changes during old age. RT-PCR was used to examine NT expression in iris and CV in 3- and 24-month-old rats. NGF and NT3 expression in iris was substantially higher compared to CV; neither target showed any alterations with age. RT-PCR for the principal NT receptors, trkA and p75, in SCG showed increased message during early postnatal life. However, during mature adulthood and old age, trkA expression remained stable while p75 declined significantly over the same period. In situ hybridization was used to examine receptor expression in subpopulations of SCG neurons identified using retrograde tracing. Eighteen to 20 h following local treatment of iris and CV with NGF, NT3 or vehicle, expression of NT receptor protein and mRNA was higher in iris- compared with CV-projecting neurons from both young and old rats. NGF and NT3 treatment had no effect on NT receptor expression in CV-projecting neurons at either age. However, similar treatment up-regulated p75 and trkA expression in iris-projecting neurons from 3-month-old, but not 24-month-old, rats. We conclude that lifelong exposure to low levels of NTs combined with impaired plasticity of NT receptor expression are predictors of neuronal vulnerability to age-related atrophy.     

Combined method of fluorescence tracer technique and PAP immunohistochemistry for discrimination of the transplanted cells

Summary The retrograde fluorescence tracer, True Blue (TB), was injected into the forebrain septal area of neonatal rats. After 3 to 6 days the brains of these animals were carefully removed and placed in ice-cold sterilized physiological saline containing 1% glucose. Under the surgical microscope, one or two pairs of mesencephalic tissue samples, each containing a dorsal raphe nucleus, were punched out and transplanted into the third ventricle of a 5,6-DHT-pretreated adult rat. One month after transplantation, all animals were perfused and their brains sectioned using a cryostat. The sections were examined using a fluorescence microscope, and then processed for serotonin immunohistochemistry. The grafts were found to be successfully implanted and connected with the middle portion of the third ventricle. Four types of neurons, i.e., TB-labeled, serotonin-labeled, both TB-and serotonin-labeled, and non-labeled neurons, were detected in the grafts. This double-labeling method is considered to be a useful technique in characterizing the neurons in grafts which consist of a heterogeneous cell population.Supporied by grants from the Ministry of Education, Science and Culture of Japan     

Time course of angiogenesis in solid pineal autografts

Angiogenesis and reperfusion of blood vessels were analysed qualitatively, at the light- and electron-microscopical levels, in solid pineal autografts placed intracerebrally in adult rats (post-transplantation survival times: 1, 3, 7, 10, 14 and 28 days). Reperfusion of blood vessels was studied in sections from immersion-fixed brains incubated to demonstrate the endogenous peroxidase activity of erythrocytes within the lumen of blood vessels. The possible presence of the blood-brain barrier (BBB) within the grafts was also investigated by injecting native horseradish peroxidase (HRP) intravenously into the rats. Angiogenesis, the morphological and functional properties of blood vessels vascularizing the grafts and the survival of pineal tissue were analysed ultrastructurally following transplantation. Revascularization of pineal autografts placed into the adult host central nervous system occurred very slowly, requiring 7–10 days to establish anastomoses between graft and host blood vessels. During this process, signs of angiogenesis in pineal and cerebral capillaries were evident, suggesting that both contributed to graft revascularization. Morphological and functional studies with HRP revealed that, following transplantation, blood vessels at the graft-host interface or within pineal autografts maintained their morphological and functional properties: they were fenestrated and did not present a BBB to blood-borne peroxidase. Thus, after grafting, the presence or absence of the BBB is graft-determined. Revascularized pineal tissue showed good survival and pinealocytes revealed structural features of active secretory cells.     

Evidence for a physiological role of nerve growth factor in the central nervous system of neonatal rats

Forebrain cholinergic neurons have been shown to respond in vivo to administration of nerve growth factor (NGF) with a prominent and selective increase of choline acetyltransferase (ChAT) activity. This has suggested that NGF can act as a trophic factor for these neurons. To test this hypothesis directly, anti-NGF antibodies (and their Fab fragments) were intracerebroventricularly injected into neonatal rats to neutralize endogenously occurring NGF. The anti-NGF antibody administration produced a decrease of ChAT activity in the hippocampus, septal area, cortex, and striatum of rat pups. This finding was substantiated by a concomitant decrease of immunopositive staining for ChAT in the septal area. These effects indicate that the occurrence of endogenous NGF in the CNS is physiologically relevant for regulating the function of forebrain cholinergic neurons.     

小鼠胚胎干细胞移植入成体大鼠脑内的区域特异性存活与分化

全能区域非特异性的胚胎干细胞是研究成体不同脑区控制干细胞分化能力的十分有力的工具。胚胎干细胞源性神经前体细胞移植入成体脑后可分化为功能性神经元,但是未分化的胚胎干细胞在成体脑内各个部位的存活、生长与分化的潜能差异尚不清楚。本文旨在探讨成体脑组织对胚胎干细胞的影响及胚胎干细胞在成体脑内的一系列行为。将少量转绿色荧光蛋白未分化的小鼠胚胎干细胞移植入成体大鼠脑内不同部位,分别于移植5、14和28d后处死大鼠,进行形态学观察及免疫组化定性,以了解未分化的小鼠胚胎干细胞在大鼠脑内不同区域的存活、生长与分化。结果发现未分化的小鼠胚胎干细胞可逐步整合入受体组织并向nestin阳性神经前体细胞分化。移植细胞及其后裔在海马生长最为旺盛,而在隔区最差（P〈0．01）;移植细胞分化为神经干细胞的效率也是在海马最高,而在隔区最低（P〈0．01）。提示只有部分脑区适合胚胎干细胞及其后裔生存,并提供促进其分化的有益环境。因此,由于位置特异的微环境因子及环境因素的存在,宿主组织特性对决定中枢神经系统疾病的细胞替代疗法策略是相当重要的。     

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.     

Ultrastructure of the capillaries of nerve tissue transplants growing in the anterior chamber of the eye

Blood capillaries have been studied electron microscopically in the areas of grafts (rat embryonal hippocamp and septal cerebral parts transplanted to mature rats) containing mainly nervous, glial or connective tissue cells. Certain differences in the capillary wall structure have been revealed. In areas with a great concentration of nervous cells, the blood capillaries are characterized by a dense arrangement of cellular elements in their walls, a continuous layer of the glial end-feet, this is specific for the CNS capillaries providing the blood--brain barrier. In peripheral area of the grafts, where glial elements predominate, the capillaries have loose arrangement of the mural cellular elements, great endotheliocyte activity, thick connective tissue tunic, lack of a dense glial surrounding. These characteristics make dubious the statement whether these capillaries possess the blood--brain barrier function. In places where connective tissue cells make aggregates, the capillaries do not possess the barrier properties because of perforations and fenestrae in endothelium and interruptions of the basal membrane, absence of pericapillary glial elements. All types of the capillaries demonstrate certain signs of a high functional activity. Formation of the capillary structure depends on the surrounding tissue.     

Neurotropic effects of estrogen on the neonatal preoptic area grafted into the adult rat brain

Summary The preoptic area (POA) or cerebral cortex taken from newborn female rats were transplanted into the third ventricle of ovariectomized adult rats. From the day of transplantation, estradiol-17/ in a silastic capsule was implanted subcutaneously into host animals for 4 weeks. The POA or cerebral cortex transplants were examined at light- and electron-microscopic levels 4 weeks after transplantation. All of the POA or cortical grafts showed an appearance similar to normal neural tissue. Estrogen exposure for 4 weeks via the host induced a significant increase in the volume of the POA grafts. The neuronal population of the POA grafts exposed to estrogen was not significantly different from that of the POA grafts without estrogen treatment. However, the number of axodendritic shaft and spine synapses of the POA grafts exposed to estrogen was significantly greater than that of the POA grafts without estrogen treatment. In contrast, there was no significant difference in the volume of the cortical tissues transplanted into the brain between the control and estrogen-treated groups. These results suggest that estrogen has a stimulatory effect on the development of neuronal substrates in the intraventricular POA graft, increasing its volume and synaptic population.     

Monitoring maturation of neural stem cell grafts within a host microenvironment

Neural stem cells (NSC) act as a versatile tool for neuronal cell replacement strategies to treat neurodegenerative disorders in which functional neurorestorative mechanisms are limited. While the beneficial effects of such cell-based therapy have already been documented in terms of neurodegeneration of various origins, a neurophysiological basis for improvement in the recovery of neurological function is still not completely understood. This overview briefly describes the cumulative evidence from electrophysiological studies of NSC-derived neurons, aimed at establishing the maturation of differentiated neurons within a host microenvironment, and their integration into the host circuits, with a particular focus on the neurogenesis of NSC grafts within the post-ischemic milieu. Overwhelming evidence demonstrates that the host microenvironment largely regulates the lineage of NSC grafts. This regulatory role, as yet underestimated, raises possibilities for the favoured maturation of a subset of neural phenotypes in order to gain timely remodelling of the impaired brain tissue and amplify the therapeutic effects of NSC-based therapy for recovery of neurological function.     

Blood supply of the bed nucleus of the stria terminalis in rat

Summary The topographical distribution of the blood vessels in the bed nucleus of the stria terminalis (NIST) has been mapped in rats. Arteries and veins were visualized in red and blue by using a double-ink perfusion technique. Arteries supplying the NIST arise from the anterior cerebral artery directly or through the anterior communicating and interhemispheric arteries. Only a few, dorsal branches derive from the medial cerebral artery through thalamostriatal arteries. According to their terminal branches, NIST arteries can be divided into five groups: medial, ventral, lateral, septal and dorsal, which have only a relatively small overlap in their territories. About 90% of veins from the NIST drain into the major basal veins. Medial branches run into the perioptic and interhemispheric veins, while the ventral branches and the large lateral vein drain directly into the anterior cerebral vein. A small proportion of NIST veins run dorsalward into the vena cerebri magna via thalamostriatal veins.     

Axons of Xenopus neural tube respond to reversals of neural tube orientation

Axonal trajectories of the Kolmer-Agduhr (KA) neurons of Xenopus embryos, were observed after anterior-posterior (A-P) inversions of neural tube grafts to determine whether KA axons follow cell-inherent directional cues, cues from their immediate environment, or rostrocaudal signals from the embryo. KA axons form one of the earliest ascending spinal pathways in Xenopus and are visible in the lateral marginal zone of whole mounts processed for GABA immunoreactivity. Grafts were made at trunk levels at stages 22–24, 3–5 h before the first KA neurons were detectable and prior to axonal outgrowth. Embryos were fixed and immunostained 6–36 h later. KA trajectories within and adjacent to reversed grafts were compared to those of nonrotated control grafts and to neural tube lengths comparable in position and in length in unoperated embryos. Most KA axons within rotated grafts followed the graft's orientation. However, others changed direction, taking novel routes including turning to conform to the orientation of the host embryo. Reorientations were most common near the posterior host/graft interface. Some host KA cells also reoriented, always within a few hundred microns of the graft interface. Taken together, these growth patterns show that most KA axons within the grafts grow normally with respect to the original polarity of the graft neural tube and maintain that direction even into tissue of opposite polarity, suggesting that their routes are mainly determined by cell-intrinsic and/or local tissue factors. However, the reorientation of many other axons, particularly near graft seams, implies that KA axons can respond to local fluctuations in directional or segment identity signals generated in both host and graft after this perturbation. © 1996 John Wiley & Sons, Inc.