Christine B. Jaeger

Long-term cross-species brain transplantation of a polymer-encapsulated dopamine-secreting cell line

Cross-species transplantation of dopamine-releasing cell lines protected against immune rejection by a semi-permeable synthetic membrane may provide a source of neurotransmitters for the treatment of Parkinsons disease. Experiments were carried out to assess whether polymer-encapsulated PC12 cells, a catecholaminergic cell line derived from a rat pheochromocytoma, could survive in vitro as well as in vivo after implantation in the striatum of adult guinea pigs. When maintained in vitro, the encapsulated PC12 cells exhibited good survival, proliferated, and spontaneously released dopamine for at least 6 months. They also retained the capacity for depolarization-elicited dopamine release. In vivo, well-preserved tyrosine hydroxylase-positive PC12 cells were observed in capsules implanted for 4, 8, and 12 weeks. Unencapsulated PC12 cells or cells in nonintact capsules did not survive transplantation at any of these time periods. The survival of encapsulated PC12 cells transplanted across species suggests that polymer encapsulation may provide an alternative for xenotransplantation of secretory cells in the absence of systemic immunosuppression.

Behavioral recovery following intrastriatal implantation of microencapsulated PC12 cells

The motor deficits associated with Parkinsons disease may be ameliorated by intrastriatal placement of dopamine-secreting cells in a polymer capsule. Water soluble polyelectrolytes were utilized for membrane encapsulation of dopamine-secreting PC12 cells. Membrane permeability studies revealed exclusion of radiolabeled 69,000 Da albumin, whereas 30,000 Da carbonic anhydrase was able to cross the membrane. No cytolytic activity was observed following incubation of the encapsulated PC12 cells with PC12 cell-directed antiserum and fresh complement. In vitro, dopamine release and the surface area of intact cells per microcapsule, reached a plateau at 4 weeks that was maintained for at least 12 weeks. Viable PC12 cells were observed in microcapsules implanted for 4 and 8 weeks in nonlesioned guinea pig striata. The behavioral effect of intrastriatal dopamine release from microencapsulated PC12 cells was evaluated in the 6-hydroxydopamine unilaterally lesioned rat model. From 1 to 4 weeks postimplantation a significant reduction in rotation behavior under apomorphine challenge was observed with PC12 cell-loaded microcapsules as compared to empty microcapsules. Tyrosine hydroxylase immunopositive PC12 cells were observed 4 weeks postimplantation in all animals exhibiting a reduction in turning behavior. Implantation of polymer-encapsulated cells may provide a means for long-term delivery of neurotransmitters and growth factors to the nervous system.

Spinal Cord Compression Injury in Guinea Pigs: Structural Changes of Endothelium and Its Perivascular Cell Associations after Blood–Brain Barrier Breakdown and Repair ☆ ☆☆

This study examines morphological changes of the blood-brain barrier (BBB) after spinal cord compression. The lowest thoracic segment (T13) of female guinea pigs was injured and the BBB was tested from 7 days to 5.5 months postinjury using intravenously injected horseradish peroxidase (HRP) as a tracer. Tracer leakage in the injured segment was verified with the light microscope and the fine structure of capillaries was examined. Diffuse tissue staining was observed at T13 up to 2 weeks following injury. A leaky BBB correlated with expected changes in the fine structure of endothelial cell junctions. These were predominantly nonoverlapping cell junctions which, in many instances, were separated by clefts between adjacent cells. At early survival times, numerous capillary profiles with juxtaposed astrocyte foot processes were noted in addition to altered cell associations. Complete sealing of the BBB against interstitial HRP leakage was not observed until 17 days postinjury. After the first week, some of the endothelial cells were contacted by macrophages, processes of perivascular microglia, and processes of swollen and degenerating astrocytes. Perivascular spaces varied in extent and contained amorphous deposits of extracellular materials in addition to supernumerary layers of basal lamina. The early changes were followed by profound tissue restructuring due to loss of both neurons and glia. At longer survival times the BBB to HRP repaired. Endothelial cells formed complex overlapping junctions with zonulae occludentes. Most of the capillaries in the injured segment were no longer in direct contact with astrocyte foot processes, although reactive astrocytes constituted the predominant cell type in the remaining gray matter. Substantial expansion of perivascular spaces was evident. The cytoplasm of endothelial cells had numerous pinocytotic vesicles. Perivascular spaces contained layers of assembled collagen arranged perpendicularly to each other in addition to amorphous matrix materials. The findings suggest that decoupling of astrocyte foot processes from endothelial cell surfaces does not prevent reformation of tight junctions. It remains to be examined what effects the larger perivascular spaces, extracellular matrix deposits, and changes of cell associations may have on transport systems and ionic buffering. The data are relevant for estimating an opportune time for application of barrier-impermeable drugs to the lesion area.

Polymer-encapsulated PC12 cells: long-term survival and associated reduction in lesion-induced rotational behavior.

Intrastriatal implantation of a dopaminergic cell line surrounded by a permeable, thermoplastic membrane was investigated as a method of long-term dopamine (DA) delivery within the central nervous system (CNS). An increase in DA release from PC12 cell-loaded capsules maintained in vitro was associated with an increase in mitotic activity of the encapsulated cell line. A significant reduction in apomorphine-induced rotational behavior was observed after PC12 cell-containing capsules were implanted into unilaterally 6-hydroxydopamine (6-OHDA) lesioned rats, which was sustained for 24 wk. Four wk after implantation, micro-dialysis studies revealed the presence of DA near PC12 cell-containing capsules, which was comparable to extracellular striatal levels of unlesioned controls. Extracellular striatal DA was undetectable by microdialysis in lesioned animals near empty polymer capsules. Histological analysis after 24 wk in vivo demonstrated that encapsulated PC12 cells survived, continued to express tyrosine hydroxylase, and that encapsulation prevented tumorigenesis. The data suggested that the release of a diffusible substance, most likely DA, from an implant is sufficient to exert a long-term functional influence upon 6-OHDA unilaterally lesioned rats and that capsules containing DA-secreting cells may be an effective method of long-term DA delivery in the CNS.

Mammalian Cortical Astrocytes Align Themselves in a Physiological Voltage Gradient

Astrocytes obtained from primary cultures of newborn rat cerebral cortex show a marked structural rearrangement to weak (50-500 mV/mm) applied voltage gradients. Astrocytes reorient their processes so that the cells are aligned perpendicular to the voltage gradient. At field strengths of 100 mV/mm or greater, this realignment occurs in over 90% of the cell population. Furthermore, these magnitudes of electric fields completely eliminate any parallel alignments originally observed prior to application of the voltage. Realignment usually occurs by a withdrawal, followed by an extension, of cell processes. These responses occur at voltage gradients within the physiological range that naturally exist across the neural tube during early development. We suggest the possibility that architectural arrangements of developing glia and, subsequently, neurons may be regulated by endogenous transepithelial potentials that exist across embryonic neuroepithelium.

Repair of the blood-brain barrier following implantation of polymer capsules

Past studies of polymer-encapsulated cell lines implanted in the brain indicated their usefulness for transmitter replacement therapy in animal models. Such grafts may have potentially important clinical applications, but their placement into neural parenchyma may cause a traumatic injury resulting in a leaky blood-brain barrier around the implant. This study investigated whether or not injury repair and reformation of the barrier takes place near a polymer capsule implanted in the brain of Sprague-Dawley rats. The two methods used for detection of a leaky barrier were immunocytochemical localization of extravasated serum albumin and circulating Evans blue that binds to serum albumin. Immunocytochemical staining for glial filament protein provided a measure for evaluating injury associated gliosis. Polymer capsules implanted for 10, 16 and 18 days were surrounded by microvessels that leaked detectable quantities of serum albumin into interstitial spaces and, by secondary uptake, into some nearby neurons and reactive astrocytes. Reactive astroglia were observed within the outer regions of the capsule wall and in the near vicinity of the implant after these early survival times. In contrast, at post-implantation times of 46 and 54 days, serum albumin was no longer detected in the neural parenchyma near the macrocapsules and only few reactive astrocytes remained. These findings show that polymer capsules implanted within the cerebrum permit (a) reformation of the blood-brain barrier and (b) occurrence of repair processes that lead to minimal deposition of reactive astroglia near the implanted polymer capsule.

Growth of tumour cell lines in polymer capsules: ultrastructure of encapsulated PC12 cells

SummaryRecent studies indicate that polymer-encapsulated PC12 cells release sufficient amounts of dopamine to significantly alter behavioural paradigms in animals with unilateral lesions of dopaminergic midbrain neurons. Because cell fine structure provides a useful measure for assessment of storage function, exocytosis, metabolism, cell activity and cell viability, we examined the ultrastructure of PC12 cells grown in semi-permeable polymer capsules maintainedin vitro or implanted into the forebrain of rats or guinea pigs. Encapsulated PC12 cells remained viable and continued to divide for the entire evaluation period of six months. Overall morphologies of encapsulated PC12 cells were similar in both environments and they resembled PC12 cells grown in monolayer cultures. In short-term cultures, encapsulated PC12 cells typically contained abundant quantities of chromaffin cell-like granules. The encapsulated cells had initially abundant microvilli on their surfaces which decline in frequency over time. After long-term enclosure for ten weeks or more, fewer secretory granules were detected in the cytoplasm of cells in capsules culturedin vitro and in brain-implated capsules. Some cells in implanted capsules had long slender filipodia that were not present on PC12 cells in cultured capsules. The morphological changes of PC12 cells may correlate with altered growth conditions such as serum and oxygen concentrations, the presence or absence of growth factors in different environments, and with changes of cell interactions related to cell densities and build up of debris within the capsules over time. Since dopaminergic PC12 pheochromocytoma cells remain viable in semi-permeable polymer capsules for at least six months, such ‘cell-capsules’ could provide an alternative to dopamine-secreting embryonic neural grafts in dopamine replacement therapies.

The mERG1a channel modulates skeletal muscle MuRF1, but not MAFbx, expression

Introduction: We investigated the mechanism by which the MERG1a K+ channel increases ubiquitin proteasome proteolysis (UPP). Methods: Hindlimb suspension and electro‐transfer of Merg1a cDNA into mouse gastrocnemius muscles induced atrophy. Results: Atrophic gastrocnemius muscles of hindlimb‐suspended mice express Merg1a, Murf1, and Mafbx genes. Electrotransfer of Merg1a significantly decreases muscle fiber size (12.6%) and increases UPP E3 ligase Murf1 mRNA (2.1‐fold) and protein (23.7%), but does not affect Mafbx E3 ligase expression. Neither Merg1a‐induced decreased fiber size nor Merg1a‐induced increased Murf1 expression is curtailed significantly by coexpression of inactive HR‐Foxo3a, a gene encoding a transcription factor known to induce Mafbx expression. Conclusions: The MERG1a K+ channel significantly increases expression of Murf1, but not Mafbx. We explored this expression pattern by expressing inactive Foxo3a and showing that it is not involved in MERG1a‐mediated expression of Murf1. These findings suggest that MERG1a may not modulate Murf1 expression through the AKT/FOXO pathway. Muscle Nerve 49:378–388, 2014

The Ubr2 gene is expressed in skeletal muscle atrophying as a result of hind limb suspension, but not Merg1a expression alone

Skeletal muscle (SKM) atrophy is a potentially debilitating condition induced by muscle disuse, denervation, many disease states, and aging. The ubiquitin proteasome pathway (UPP) contributes greatly to the protein loss suffered in muscle atrophy. The MERG1a K+ channel is known to induce UPP activity and atrophy in SKM. It has been further demonstrated that the mouse ether-a-gogo-related gene (Merg)1a channel modulates expression of MURF1, an E3 ligase component of the UPP, while it does not affect expression of the UPP E3 ligase Mafbx/ATROGIN1. Because the UBR2 E3 ligase is known to participate in SKM atrophy, we have investigated the effect of Merg1a expression and hind limb suspension on Ubr2 expression. Here, we report that hind limb suspension results in a significant 25.6% decrease in mouse gastrocnemius muscle fiber cross sectional area (CSA) and that electro-transfer of Merg1a alone into gastrocnemius muscles yields a 15.3% decrease in CSA after 7 days. More interestingly, we discovered that hind limb suspension caused a significant 8-fold increase in Merg1a expression and a significant 4.7-fold increase in Ubr2 transcript after 4 days, while electro-transfer of Merg1a into gastrocnemius muscles resulted in a significant 6.2-fold increase in Merg1a transcript after 4 days but had no effect on Ubr2 expression. In summary, the MERG1a K+ channel, known to induce atrophy and MURF1 E3 ligase expression, does not affect UBR2 E3 ligase transcript levels. Therefore, to date, the MERG1a channel’s contribution to UPP activity appears mainly to be through up-regulation of Murf1 gene expression.