Stereological analysis of synaptogenesis in the molecular layer of piriform cortex in the prenatal rat.

The development of synapses in the molecular layer of the rat piriform cortex was studied at embryonic days 15, 17, 19, and 21. The present study has sought to extend past studies of synaptogenesis by identifying not only changes in numbers of synapses, but also changes in numbers of potential precursors of synapses. A stereological method (Cruz-Orive, '80) was used to make volumetric estimations of the numbers of synapses, axonal puncta, vesicle-associated puncta, and unapposed postsynaptic specializations. This stereological method was preferred to other morphometric methods because it is not influenced by changes in the size, shape, or orientation of the structures of interest. This was considered important since such changes might be expected during development. Large numbers of unapposed axonal specializations (axonal puncta and vesicle-associated puncta) were found in all three sublaminae (lateral olfactory tract, Ia, and Ib) at all ages. The numerical density (number per unit volume of neuropil) and relative frequency of these structures changed significantly with time. In all three sublaminae, these changes were associated with changes in the number of synapses, although the numerical density and relative proportions varied between the sublaminae. These results suggested that axonal puncta could accumulate vesicles, thus becoming vesicle-associated puncta, and that vesicle-associated puncta could contact dendrites, thus forming synapses. In contrast, the numerical density of lone postsynaptic specializations remained low and no significant changes in their relative proportion in the population were found. This suggested that although lone postsynaptic sites were observed, they did not appear to play a major role in synaptogenesis in this region of the cortex. In addition to documenting developmental differences between the three sublaminae in the molecular layer, the results support a synaptogenic hypothesis in which the axon can form surface specializations that appear to be involved in synaptogenesis, independent of direct dendritic contact.

Application of quality assurance practices in processing cells and tissues for transplantation.

Attention to issues of quality assurance from the early stages of development of experimental cell therapies provides a margin of safety for recipients. Adherence to minimum standards of practice at acquisition, processing, storage, and implantation ensures not only this baseline safety factor for patients, but also provides a baseline for comparative evaluations between different studies or different banks. This paper describes the basic components of a quality assurance program tailored to laboratories and facilities that collect, process, or distribute human cells and tissues for transplantation. These components include policy and procedure manuals, donor screening practices, processing procedures evaluation and control, training and education programs, auditing and investigation roles, responsibility for release of grafts, and recordkeeping and traceability requirements. References and resources for detailed information related to good manufacturing practices and good clinical and laboratory practices are provided. Standards, regulations, and current legislation specifically related to human cells and tissues intended for transplantation are also discussed.

Effect of storage and preservation methods on viability in transplantable human skin allografts.

This study compared the metabolic activity of fresh skin samples to that of cadaver human skin allografts processed and stored by current tissue banking methods. We chose to use two metabolic assays as surrogate measures for viability in these grafts. Skin allografts stored either in liquid media at 4 degrees C for varying periods of time or stored by cryopreservation were quantitatively assessed for viability by tetrazolium reduction and oxygen consumption assays. These measurements were compared to viability assessments of fresh autograft skin. Human cadaver skin grafts, after procurement and just prior to further tissue bank processing, exhibited approximately 60% of the metabolic activity found in fresh skin samples obtained from living surgical donors. If allowed an overnight (18-24 h) incubation period at 37 degrees C, cadaver samples showed a recovery of their metabolic activity to 95% of that found in the autograft skin samples. When stored in liquid media at 4 degrees C, the cadaver skin declined steadily in cellular metabolic activity, arriving in less than 5 days storage at a measurement below that of cryopreserved skin. The cryopreserved skin was measured both immediately after thawing and dilution of cryoprotectant, as well as after equilibration and overnight incubation. Skin cryopreserved with dimethylsulfoxide Me(2)SO retained higher viability than glycerol cryopreserved skin. Residual concentrations of cryoprotectants were determined following typical recommendations for thawing and diluting skin allografts. The implications of these findings for transplantation and tissue banking are discussed.

Fetal tissue banking for transplantation: characteristics of the donor population and considerations for donor and tissue screening.

We initiated this study to evaluate the suitability for therapeutic use in transplantation of tissues obtained from human abortuses. We have developed protocols for the collection, handling and preservation of hepatic stem cells from electively aborted embryos and have developed methods for assessment of the cells so derived and processed. In this paper we present our findings regarding screening of potential donors, acquisition of fetal tissues, and assessment of the tissues for potentially infectious contaminants. We assess the suitability of the tissue donors according to current standards used for donors of commonly transplanted tissues (e.g., bone grafts, skin grafts and heart valves) and present data regarding the real availability of tissues from elective abortion procedures that would meet those standard tissue banking criteria.We specifically evaluated the donor's willingness to provide a blood sample for testing, conducted a detailed interview similar to those used for typical organ and tissue donors, and assessed the type and incidence of contamination in collected tissues. We find that although many women are willing to consent to use of the tissues for transplantation, attrition from the study for various reasons results in few fetal organs ultimately realistically available for transplantation. Typical reasons for attrition include: unwillingness to have a blood sample drawn or tested, positive serology results, social/medical high risk factors for acquisition of transmissible disease, no identifiable organs available, and unacceptable microbial contamination. Thus, although it might seem that due to the numbers of abortions performed annually, that there would be substantial numbers of suitable tissues available, only a small proportion are truly suitable for transplantation.

Fetal tissue banking: standards and regulatory issues.

There has been a long-standing interest in collection and study of fetal tissues for the purpose of understanding normal ontogeny, as well as aberrant processes in development. As the unique features and capabilities of fetal tissues have become elucidated, it is evident that fetal tissues could potentially be used to ameliorate adult degenerative diseases through transplantation. Indeed, there has been significant work surrounding the transplantation of fetal-derived hematopoietic stem cells, islet cells, and central nervous system cells. Many involved with collection and transplantation of fetal tissues seem poised on the edge of the federal regulatory playing field. This article discusses the history of regulations related to cellular and tissue-derived products and current regulatory issues facing cell and tissue banks in the United States from the tissue bank perspective and focuses on these issues as they relate to the use of cells derived from fetal tissue.

Expression of calcium/calmodulin-dependent protein kinase in relation to dopamine islands and synaptic maturation in the cat striatum.

During pre- and postnatal development the dopamine-containing nigrostriatal afferents of the striatum are arranged as a conspicuous series of patches (the "dopamine islands"). The development of this dopamine island system, which metamorphoses in early postnatal life to the striosomal architecture of the adult, has recently received considerable attention, but the factors initiating and influencing maturation of this architecture are largely unknown. In an attempt to clarify the relationships between the onset of clustering of dopamine-containing afferents, the grouping of neurons within future striosomes and the maturation of synapses in the striatum, we compared the initial prenatal appearance and subsequent development of immunohistochemical markers for the dopamine-containing innervation [tyrosine hydroxylase (TH)-like immunoreactivity], for synaptic vesicles (SV48-like immunoreactivity), and for a phosphorylation-related enzyme Ca2+/calmodulin-dependent protein kinase type II (CaM kinase II-like immunoreactivity) that is expressed in virtually all striatal neurons by adulthood. Here we present evidence that during striatal ontogeny, both neurons and neuropil expressing CaM kinase II-like immunoreactivity and SV48-positive terminals form discrete patches that are in register with dopamine islands. It is CaM kinase II-positive elements, however, rather than the TH-positive island fibers (or SV48-positive synapses), that initially form overt clusters. Dopamine-containing fibers begin to innervate the striatal anlage just prior to embryonic day (E) 32. Their distribution follows the general lateral to medial developmental gradient characteristic of the striatum but is not yet distinctly islandic. At this time, CaM kinase II-like immunoreactivity was very weak or not present at all and SV48-like immunoreactivity was undetectable. By E36, CaM kinase II-positive neurons are visible in discrete patches of immunopositive neuropil, but only faint inhomogeneities are detectable in the distribution of TH-positive fibers and scarcely any SV48-like immunoreactivity can be seen. By E45, all 3 markers are focused in typical islandic patterns, and they remain so into the early postnatal period. These observations suggest a developmental sequence in which dopamine-containing fibers invade the striatal anlage prior to forming distinct islandic foci and prior to the maturational events signaled by the production of CaM kinase II within the neurons and neuropil of future striosomes.(ABSTRACT TRUNCATED AT 400 WORDS)

Independent development of presynaptic specializations in olfactory cortex of the fetal rat.

Electron microscopy was used to study synaptogenesis in prepyriform cortex of fetal rat pups during early stages of synapse formation. Of special interest is the frequent occurrence of unapposed, developing synaptic specializations in axon and growth cone profiles. The location and morphology of the unapposed specializations suggests that they are presynaptic in nature. These presumably immature presynaptic specializations are found in the lateral olfactory tract and subjacent cortex. Intermediate forms between uncontacted presynaptic specializations and definitive synapses suggest a synaptogenic sequence in which initial development of an immature presynaptic specialization begins without apposition of a postsynaptic element at that location. This implies that initiation of presynaptic development is not dependent upon postsynaptic contact and also raises the question of whether synaptic contacts could be established via presynaptic induction of postsynaptic formation.