Growth of brain tissue grafts is dependent upon host age

Growth of grafts of cortex cerebri, hippocampus, septum and cerebellum in oculo were significantly reduced in 16--17-month-old hosts as compared to growth in 3-month-old and 1.5-month-old rat hosts. (Host age is given as the age of the recipients at the time of grafting.) This growth difference was less pronounced in locus coeruleus grafts. The vascular network (as observed with laminin immunofluorescence) in cortex cerebri, hippocampus, cerebellum and septum grafts in 16--17-month-old hosts was abnormal with few thick-walled vessels in clusters as compared to the more 'normal' vascularization found in 1.5-month-old hosts with a high number of thin-walled blood vessels evenly distributed throughout the grafts. Grafts in the oldests hosts were markedly more gliotic than grafts in 1.5- and 3-month-old hosts as evaluated using immunofluorescence with antibodies against glial fibrillary acidic protein. Neurofilament immunoreactivity in the grafts seemed not to be influenced by host age. When a second cortex cerebri or hippocampus graft was placed into contact with a previously grafted locus coeruleus graft, the second graft grew less well in 16--17-month-old hosts as compared to 1.5-month-old hosts. When cortex cerebri was added to a previously grafted cortex cerebri graft, the second graft in both 16--17- and 3-month-old hosts grew to larger sizes than the corresponding single cortex grafts, although the growth differences between the two groups of hosts described above were still maintained. Thus, cortex grafts in 16--17-month-old hosts still have the ability to become trophically stimulated. The vascularization of the second graft in both groups was almost normalized and the gliotic reaction was less pronounced in the second grafts in both groups as compared to the single cortex grafts. In conclusion, the present results indicate that host age affects growth and morphology of intraocular single grafts from several brain regions. Using double grafts of cortex cerebri it was shown that grafts in 16-17-month-old hosts still had the capacity to become trophically stimulated. Data on brain transplants in older hosts are important in view of clinical possibilities to use transplantation strategies to counteract the symptoms of neurodegenerative diseases, which usually occur in old patients.     

Construction of chimeric thymuses in the mouse fetus by in utero surgery

Normal thymuses develop in the coelomic cavity of mice following grafts carried out in utero into embryonic day 16 to 17 fetuses of the third branchial arch region removed from 10-day embryos. The recipients were killed at various times (up to 29 days after birth) and the thymic lobes which developed from the graft were recovered. Microscopical examination and immunocytochemistry revealed that these thymuses developed normally whether the grafts involved syngeneic or allogeneic (between BALB/c and C3H) strain combinations. Two major populations of class II-expressing cells could be recognized by the Ia allotype they expressed: epithelial cells of donor type and accessory cells of host origin.     

Age-related changes induced by tunicamycin in wheat germ agglutinin binding to chick embryo fibroblasts

The specific roles of N-acetylglucosaminyl and sialyl residues were investigated in the binding of wheat germ agglutinin (WGA) to fibroblasts from 8- and 16-day chick embryos. Cells from the 8-day embryos exhibited two classes of WGA binding site, whereas fibroblasts from 16-day embryos only displayed one. Neuraminidase treatment of fibroblasts from 8-day embryos raised the number of WGA binding sites with a high affinity constant and reduced the number of sites with a low affinity constant. In 16-day cells, neuraminidase treatment reduced the number of WGA binding sites. The data presented here suggest that WGA binds to cell-surface glycoproteins containing sialic acid residues. This tendency was more marked in 16-day than in 8-day cells and corresponded to the finding that neuraminidase released more sialic acid from the surface of 16-day cells than of 8-day cells. Glycosylation of cell-surface N-linked glycoproteins did not seem essential in determining the WGA binding capacity, as shown by the effect of tunicamycin on fibroblasts from both 8- and 16-day embryos. In the absence of N-glycosylated binding sites, WGA bound to O-glycosylated structures and the older tunicamycin-treated embryo cells exhibited a larger number of WGA binding sites than the younger tunicamycin-treated cells, in relation to the increase of the amount of O-glycosylated structures during embryo development.     

Can neurografts from mice with chromosome 16 trisomy serve as a model of Alzheimer disease?

Basal forebrain regions were transplanted from mouse embryos with chromosome 16 trisomy into the lateral brain ventricle of healthy adult mice. After 1–12 months, the grafts contained immunoreactive choline- and GABAergic neurons and astroglial cells. Quantitative analysis revealed no neurodegenerative changes in these grafts compared to diploid ones. Pronounced infiltration with T and B cells and activation of micro- and astroglia were found in the grafts with chromosome 16 trisomy and in control specimen. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 128, No. 11, pp. 559–564, November, 1999     

Development of functional synaptic connections in the auditory system visualized with optical recording: afferent-evoked activity is present from early stages

A comprehensive survey of auditory network formation was performed in the brain stem of the chicken embryo using voltage-sensitive dye recording. Intact medulla/brain stem preparations with the auditory branch of the eighth nerve attached were dissected from 5.5- to 8-day chicken embryos, and responses evoked by nerve stimulation were recorded optically. In the medulla of 7- and 8-day embryos, we identified four response areas, corresponding to ipsilateral Nucleus magnocellularis (NM) and Nucleus angularis (NA), which receive the auditory afferents, and ipsi- and contralateral Nucleus laminaris (NL), which receive projections from NM. The optical responses consisted of a fast spikelike signal followed by a long-lasting slow signal, which reflected the sodium-dependent action potential and glutamatergic excitatory postsynaptic potential (EPSP), respectively. In NM, NA, and NL, the EPSP-related slow optical signals were detected from some 6-day and all 7- and 8-day preparations, indicating that functional synaptic connectivity in these nuclei arises by the 7-day stage. In the pons of 7- and 8-day embryos, we identified two additional response areas, which evidently correspond to ipsi- and contralateral Nucleus lemnisci lateralis (NLL), the higher-order nuclei of the auditory pathway. Furthermore, we detected optical responses from the contralateral cerebellum, which possibly correspond to transient projections observed only during embryogenesis. The present study demonstrates that functional auditory circuits are established in the chicken embryo at stages earlier than previously reported. We discuss the possible role of afferent-evoked activity with reference to auditory neural network formation.     

Tim-1-Fc suppresses chronic cardiac allograft rejection and vasculopathy by reducing IL-17 production

Previously, we demonstrated that Tim-1-Fc prevents acute cardiac graft rejection by inhibiting Th1 response. In the present report, we tackled the impact of Tim-1-Fc on Th17 cells in a model of cardiac chronic rejection. Administration of Tim-1-Fc did not result in a detectable impact on innate immunity and regulatory T cells, while it provided protection for Bm12-derive cardiac grafts against chronic rejection in B6 recipients, as manifested by the reduction of inflammatory infiltration along with less severity of vasculopathy. Studies in T-bet^-/- recipients by implanting Bm12-derived cardiac grafts further revealed that Tim-1-Fc significantly protected cardiac grafts from chronic rejection along with attenuated production of IL-17 producing T cells. Depletion of CD4 and CD8 T cells or blockade of IL-17 in T-bet^-/- recipients demonstrated that Tim-1-Fc selectively suppresses Th17 differentiation along with attenuated IL-17 secretion. Together, our data suggest that Tim-1-Fc protects cardiac grafts from chronic rejection by suppressing CD4 Th17 development and functionality. Therefore, Tim-1-Fc might be a potential immunosuppressive agent in the setting of cardiac transplantation.     

Hydroxyproline and hydroxylysine in different tissues of human embryos.

The collagen content in various tissues of human embryos was studied at four different steps of the maturation process. The umbilical cord of a 16-17 week old embryo was found to be active in collagen biosynthesis, Tibia, articular cartilage and skin showed of a peak of total hydroxyproline in the 16-17th week, decreasing later on, while the hydroxyproline decreased in umbilical cord from the 15-16th week to the 24th week. Hydroxylysine followed the hydroxyproline changes in articular cartilage and umbilical cord.     

Ability of neural crest cells from the embryonic chick to differentiate into cartilage before their migration away from the neural tube

Whether neural crest cells from the avian embryo are determined for chondrogenesis before they begin their migration away from the neural tube (i.e., before H. H. stages 8.5-9) was investigated by establishing neural folds from embryos of H. H. stages 5-11 either in organ culture, or as grafts to the chorioallantoic membranes of host embryos. Cartilage differentiated from neural folds taken from embryos of H. H. stages 5-7 but not from those taken from older embryos. This stage specific pattern was reversed when the tissue adjacent to the neural tube was grafted to the chorioallantoic membrane. Cartilage only formed from tissues isolated later than H. H. stage 8; i.e., when these adjacent tissues contain neural crest cells. We concluded that neural crest cells are determined for chondrogenesis while still in the neural tube and before their migration to the face and head. This is in contrast to the situation in the only other group which has been examined, the urodele amphibians.     

Optical approaches to functional organization of glossopharyngeal and vagal motor nuclei in the embryonic chick hindbrain

We investigated the functional organization of the glossopharyngeal and vagal motor nuclei during embryogenesis using multiple-site optical recording with a fast voltage-sensitive dye. Intact brain stem preparations with glossopharyngeal and vagus nerves were dissected from 4- to 8-day-old chick embryos. Electrical responses evoked by glossopharyngeal/vagus nerve stimulation were optically recorded from many loci of the stained preparations. In 4- to 6-day-old preparations, action potential-related fast spikelike signals were detected from the nucleus of the glossopharyngeal nerve and the dorsal motor nucleus of the vagus nerve. Contour line maps of the signal amplitude showed multiple-peak patterns, suggesting that the neurons and/or their activity were not uniformly distributed within the nuclei at early developmental stages. As development proceeded from 4 to 6 days, the peaks fused with each other and the number of peaks decreased gradually. In most 7- and 8-day-old preparations, only a single peak was identified in the nuclei, and the distribution of the signal amplitude formed a layered pattern surrounding the peak-signal area. These results suggest that functional organization of the motor nuclei in the embryonic hindbrain changes dynamically with development, resulting in a rearrangement of functional nuclear cores from multiple-peaks to a single peak.     

Cellular characterization of blastocysts derived from rabbit 4-, 8- and 16-cell embryos and isolated blastomeres cultured in vitro

The purpose of this study was to investigate the developmental potential of isolated rabbit blastomeres under various culture conditions to gain insight into their ability to form the two cell lineages of a viable blastocyst. Intact embryos at the 4-cell, 8-cell, 16-cell stages and blastomeres isolated from 4-, 8- and 16-cell rabbit embryos (1/4, 1/8 or 1/16 blastomeres respectively) were cultured in drops of one of three different media, each supplemented with either fetal calf serum (FCS), bovine serum albumin (BSA) or polyvinyl alcohol (PVA). The effects of the extracellular matrix fibronectin (FN) on the development of isolated rabbit blastomeres were also investigated. Supplementation of the medium with FCS yielded a higher (P < 0.05) proportion of blastocysts than BSA or PVA, predominantly from 1/4 blastomeres. No major differences were found between the three basic culture media. In 1/4, 1/8 or 1/16 blastomeres, blastocyst formation rates were greater (P < 0.05) in groups cultured in matrix-free (54.5, 59.6 and 54.6% respectively) than in FN-coated groups (35.4, 46.0 and 26.1% respectively). Only in blastocysts derived from 1/4 blastomeres, were the numbers of inner cell mass (ICM) and total cells of blastocysts higher (P < 0.05) in FN-coated groups than in matrix-free groups (12.7 +/- 1.1 versus 8.5 +/- 0.7 ICM, 73.8 +/- 3. 7 versus 57.8 +/- 3.3 total cells). The percentage of blastocysts derived from single blastomeres with ICM cells decreased with increasing cell stage of the parent embryos in FN-coated (93.6, 78.3 and 44.0%, respectively) as well as matrix-free groups (96.2, 69.3 and 55.2%). In FN-coated groups, after 96 h (1/4) or 72 h (1/8 and 1/16) of culture, approximately 20-30% of blastomeres did not develop into normal blastocysts but formed sheets with 30-50 cells attached to the bottom of the dishes. These results indicate that the development of rabbit blastomeres shares important characteristics with those from mouse and domestic species and may thus aid in developing an efficient culture system for blastomeres, derived from human embryos.     

Epithelial influences on skeletogenesis in the mandible of the embryonic chick

Intact mandibular processes and the enzymatically separated mesenchymal and epithelial components of the mandible from embryonic chicks of 2.5- to 5-day incubation (Hamburger and Hamilton, '51: stages 16-25) were grown individually, either in organ culture or as grafts to the chorioallantoic membranes of host embryos. The differentiation of cultured and grafted intact mandibular processes was histologically normal, but the time of histodifferentiation differed from that in vivo. The histodifferentiation of cultured and grafted mandibular mesenchyme grown isolated from its epithelium depended upon the age of the embryo from which the mesenchyme had been obtained. Intramembranous ossification producing membrane bones of the mandible occurred in mesenchyme isolated from 4.5- to 5-day embryos (HH 24–25), but did not occur in mesenchyme isolated from younger embryos. Cartilage (Meckel's) and subperichondrial bone in the articular process of Meckel's cartilage differentiated in mesenchyme isolated from embryos of all age groups tested (HH 16–25). Mandibular mesenchyme, therefore, requires the presence of epithelium until 4.5 days of incubation if the membrane bones of the mandible are to differentiate; if epithelial influences are required for Meckel's cartilage and subperichondrial bone formation, they are not required beyond 2.5 days of incubation. Mandibular epithelium isolated from its mesenchyme became layers of squamous cells in culture; but when grafted onto the chorioallantoic membrane, the epithelium became underlain by host fibroblasts and differentiated into a stratified squamous epithelium. Mandibular epithelium, therefore, is capable of differentiation in the presence of foreign fibroblasts derived from the chorioallantoic membrane.     

Improved survival of young donor age dopamine grafts in a rat model of Parkinson's disease

In an attempt to improve the survival of implanted dopamine cells, we have readdressed the optimal embryonic donor age for dopamine grafts. In a rat model of Parkinson's disease, animals with unilateral 6-hydroxydopamine lesions of the median forebrain bundle received dopamine-rich ventral mesencephalic grafts derived from embryos of crown to rump length 4, 6, 9, or 10.5 mm (estimated embryonic age (E) 11, E12, E13 and E14 days post-coitus, respectively). Grafts derived from 4 mm embryos survived poorly, with less than 1% of the implanted dopamine cells surviving. Grafts derived from 9 mm and 10.5 mm embryos were similar to those seen in previous experiments with survival rates of 8% and 7% respectively. The best survival was seen in the group that received 6 mm grafts, which were significantly larger than all other graft groups. Mean dopamine cell survival in the 6 mm group (E12) was 36%, an extremely high survival rate for primary, untreated ventral mesencephalic grafts applied as a single placement, and more than fivefold larger than the survival rate observed in the 10.5 mm (E14) group. As E12 ventral mesencephalic tissues contain few, if any, differentiated dopamine cells we conclude that the large numbers of dopamine cells seen in the 6 mm grafts must have differentiated post-implantation. We consider the in vivo conditions which allow this differentiation to occur, and the implications for the future of clinical trials based on dopamine cell replacement therapy.     

Abnormal expression of tyrosine hydroxylase-like immunoreactivity in intraocular transplants of rat caudate nucleus

The purpose of the present study was to examine tyrosine hydroxylase (TH)-like immunoreactivity in single intraocular grafts of caudate nucleus, and in caudate grafts which were co-grafted with substantia nigra. Grafts of caudate obtained from fetal rats (crown-rump length: 15-17 mm) were placed into the anterior chamber of eye of adult female Sprague-Dawley rats, and were allowed to survive from 6 to 20 months. The host rats were then perfused with fixative and the tissue was prepared for immunocytochemistry for TH. In 7 out of 8 surviving caudate grafts, including those containing no substantia nigra, tyrosine TH-like immunoreactive (THLI) cell bodies were present. The results of our study suggest that TH is inappropriately expressed in caudate grafts placed in the anterior chamber of the eye.     

Isotransplantation of fetal mouse pancreas in experimental diabetes. Effect of gestational age and organ culture

Fetal mouse pancreatic isotransplantation was performed in CBA mice, previously made diabetic with streptozotocin. Either 17- or 12-day-old fetal tissue was used, either uncultured or after 14 days in organ culture. Grafts were placed under the renal capsule, and their effect on fasting blood glucose levels was assessed over a 10-week postgraft period. The grafts and the host pancreas were then examined by light microscopy. Uncultured 12- and 17-day fetal pancreases produced poorly developed grafts with small islets, numerous ducts, and copious adipose tissue. By contrast, if organ cultured for 14 days before transplantation, both 12- and 17-day tissue resulted in large grafts composed predominantly of well-stained islet cells, few ducts, and little connective and adipose tissue. The most functionally effective grafts were derived from cultured 17-day pancreas, and a single graft per recipient always ameliorated diabetes. By contrast, grafts from single or multiple uncultured 12-day pancreases did not function, but, after culture, grafts derived from three (but not from one) 12-day pancreas lowered blood glucose levels to almost normal levels. It is concluded that grafts of fetal mouse pancreas can function in experimental diabetes provided they are first cultured and are of adequate gestational age.     

Transplantation of brain tissue in the brain of rat. I. Growth characteristics of neocortical transplants from embryos of different ages

Differential growth of neural transplants as related to the age of the donor embryos was investigated in this study. Neocortical tissue of constant volume, obtained from embryos of 15, 16, 17, 18, 19, 20, and 21 days' gestational age, was transplanted into the cerebellum of 10-day-old rats. The fully grown transplants were analyzed quantitatively and qualitatively 90 days after transplantation. The ultimate volume of the transplants and the estimated total number of neurons in them followed a gradient in relation to the age of the donor embryos. At one extreme, the neural transplants from 15-day-old embryos grew very large, showing a 21-fold increase in size, and at the other extreme, those from 21-day-old embryos grew less than two-fold in volume. These differences were determined by the developmental history of the transplants. Neural tissue obtained from 15-day-old embryos contained predominantly neuroepithelial cells which continued to proliferate even after transplantation. This resulted in the large size of these transplants. At the other extreme, neural tissue from 21-day-old embryos contained predominantly preformed neuroblasts, and they simply differentiated after transplantation. Due to this, the transplants were small in size. Neural tissues obtained from other embryos of different gestational ages between these two extremes contained neuroepithelial cells and preformed neuroblasts in differential ratios. The number of neuroepithelial cells in the transplants and their differential proliferative activity after transplantation, and the number of neuroblasts present, determined the differential sizes of these transplants. In histological preparations, all transplants were seen to contain normal-looking and well-differentiated neurons, and normal-looking neuropil. The transplants were integrated with the host brain, in that there was neither any gap nor any scar tissue between the transplants and the host neural tissue surrounding them. Neither the transplants nor the host brains showed any pathological reaction or neoplastic growth.     

Recovery of functional deficits following early donor age ventral mesencephalic grafts in a rat model of Parkinson's disease

It has previously been reported that dopaminergic grafts derived from early donor age, embryonic age 12-day-old (E12) rat embryos produced a fivefold greater yield of dopamine neurons than those derived from conventional E14 donors. The present study addresses whether E12 grafts are able to ameliorate lesion-induced behavioral deficits to the same extent as E14 grafts. In a unilateral rat model of Parkinson's disease, animals received grafts derived from either E12 or E14 donor embryos, dispersed at four sites in the lesioned striatum. Both E12 and E14 grafts were able to induce recovery on both amphetamine and apomorphine rotation tests, and to ameliorate deficits in the cylinder, stepping test, and corridor tests, but were unable to restore function in the paw reaching task. E12 grafts were equivalent to E14 grafts in their effects on lesion-induced deficits. However, E12 grafts resulted in cell yields greater than previously reported for untreated primary tissue, with mean TH-positive cell counts in excess of 25,000 neurons, compared with E14 TH cell counts of 4000-5000 cells, representing survival rates of 75% and 12.5%, respectively, based on the expected adult complement. The equivalence of graft induced behavioral recovery between the two graft groups is attributed to a threshold number of cells, above which no further improvement is seen. Such high dopamine cell survival rates should mean that multiple, functioning grafts can be derived from a single embryonic donor, and if similar yields could be obtained from human tissues then the goal of one embryo per patient would be achieved.     

Cytogenetic analysis in a case of cancer of the male breast

Sequential chromosome banding of direct preparations from an infiltrating ductal carcinoma of the left breast of a male, aged 56 years, showed a diploid chromosome range with a mode at 44. Consistent monosomy of chromosomes #2, #3, #4, #6, #11, #15, and #17 and nullisomy of chromosomes #1, #8, and #12 were found. In addition, each cell contained 11-14 markers and 1-7 abnormal chromosomes. Altogether, 16 markers were characterized, and 2 of these involved the long arm of chromosome #1. This chromosome pattern is similar to that in diploid breast carcinomas of the female.     

Ploidy in human cleavage stage embryos after fertilization in vitro

Chromosome preparations were made from 15 cleaved human embryosin the 2- to 12-cell stage after in-vitro fertilization. Allshowed two pronuclei before the first cleavage. Twelve had atleast one diploid metaphase, while three had interphase nucleionly. There was no evidence of partheno-genetic haploid cleavage.As expected, the frequency of metaphases increased with durationof colchicine treatment: 25 and 55% of the cells reached metaphaseafter 6–13 and 16–24 hours' treatment, respectively.Cleaved embryos with ‘ideal’ blastomere numbers(2, 4 or 8) showed a considerably higher metaphase frequencythan others. For a more detailed chromosome analysis the techniquewill have to be further improved. The asynchronous cleavageof blastomeres makes optimal treatment by mitogens difficult.     

Timing of odontogenic neural crest cell migration and tooth-forming capability in mice.

The mammalian tooth develops through sequential and reciprocal interactions between cranial neural crest (CNC)- derived ectomesenchymal cells and the stomadial epithelium. Classic tissue recombination studies demonstrated that premigratory CNC cells and CNC-derived ectomesenchymal cells possess odontogenic capacity and can respond to oral epithelial signals to form a tooth, suggesting that the CNC cells contributing to odontogenic tissue are not prespecified. Here we show that, in mice, CNC cells have populated the forming first branchial arch before the 9-somite stage and continue to migrate into the arch by the 13-somite stage. Grafts of the first arch from the 10-somite embryo or earlier yielded membranous bone and cysts but no teeth after subrenal culture. However, grafts of the first arch with its dorsally adjacent tissue containing migrating neural crest cells from the same age embryos gave rise to teeth. In contrast, teeth formed in first arch grafts that do not contain migrating neural crest cells from embryos with 12 or more somites. Interestingly, the acquisition of tooth forming capability in the first arch coincides with the onset of Fgf8 expression in the oral epithelium. These results suggest that there exists a population of odontogenic neural crest cells that migrates into the first arch between the 10- and 12-somite stages. These cells either possess odontogenic potential and are able to initiate tooth development, or can respond to odontogenic signals derived from the oral epithelium to support tooth formation.     

In vitro embryotoxic effects of ethylene glycol in rats

Rat embryos of the CD strain were treated in a whole embryo culture system with either 30 or 40 microliters of ethylene glycol (EG) per milliliter of culture medium for the first 8 (0-8 hr) or second 8 (8-16 hr) hr of a 48-hr culture period. The compound was not embryolethal under these conditions but did alter growth and development. EG at 40 microliters/ml of culture medium at 8-16 hr decreased morphological score, somite number, crown-rump and head lengths, as well as DNA and protein contents. The most frequent abnormality induced by the compound was absence of yolk sac circulation; absent hindlimb bud, hypoplastic telencephalon, and lack of development of the otic and optic systems were also seen in EG-exposed embryos. Since it has been reported that rodent embryos cultured in vitro lack alcohol and aldehyde dehydrogenases that metabolize EG, present results suggest that the parent compound is capable of altering normal embryonic development when administered during a brief period or organogenesis.