bluuebunny/crossref_metadata_2025 huggingface dataset:
Repeated Intraportal Injections of Subtherapeutic Islet Cell Isografts Restore Normoglycemia in Streptozotocin-Diabetic Rats
Authors: Morsiani Eugenio , Rozga Jacek , Dellagiacoma Giovanni , Demetriou Achilles A.
Published: June 2017

Poor engraftment and consequent loss of β-cell mass could be one of the factors that are responsible for function loss after intraportal islet transplantation (Tx). Streptozotocin-diabetic rats were transplanted with syngeneic islets, which were injected into the portal vein via an indwelling catheter connected to a subcutaneous port. In Group I (n = 6), 1,000 islets were injected in a single dose into the liver. In Group II (n = 6), five doses of 200 islets were repeatedly injected over a period of 14 days, for a total of 1,000 islets. In Group III (n = 4), five decreasing doses of islets were injected over a period of 14 days, for a total of 750 islets. Nonfasting blood glucose (n-FBG) and body weight (b.wt.) were determined twice a week and an intravenous glucose tolerance test (IVGTT) was performed at 30 and 90 days. In Group I, n-FBG decreased in 2 wk from the time of first islet injection, averaging 110 ± 21.9 mg/dL at 1 mo (p < 0.05 vs. normal controls); this value was maintained throughout the 3-mo duration of the study. In Group II, n-FBG was normalized in 2 wk averaging 90.2 ± 25 mg/dL on day 12 (p = NS vs. normal controls) and 75.8 ± 14.6 mg/dL at 1 month (p = NS vs. normal controls); this value was maintained throughout the 3-mo duration of the study. In Group III, n-FBG decreased to normal values in 2 wk, averaging 77 ± 15.7 mg/dL at 1 mo (p = NS vs. normal controls), but normoglycemia was maintained for 40 days and then followed by a progressive increase. Only in Group II, KG (percent/min decline in glucose level) was not significantly different from that of normal controls (1.702 ± 0.531 at 1 mo and 1.676 ± 0.891 at 3 mo), while it was significantly lower than normal controls in both Group I and III animals. Body weight increase after Tx correlated with the number of transplanted islets and at 90 days, Group III rats showed less increase than Groups I and II (p < 0.05), while no significant differences in b.wt. were recorded between Group I and II. The findings indicate that intraportal islet Tx, injected repeatedly and in small doses, produced better metabolic effects than injection of the same total number of islets in a single dose. Copyright © 1997 Elsevier Science Inc.
Effects of Caffeine and Acetylcholine on Glucose-Stimulated Insulin Release from Islet Transplants in Mice
Authors: Shi Chun-Liang
Published: June 2017

In mouse islet grafts under the kidney capsule, the potentiating responsiveness to acetylcholine was markedly attenuated after a few weeks. The question arose as to whether transplanted islets show an decreased responsiveness to potentiators in general. The effect of caffeine on glucose-induced insulin secretion was, therefore, examined. Intrastrain transplantation was performed in NMRI and BALB/c mice, and islet grafts were removed and perifused in vitro after 3 and 12 wk. In grafts from both NMRI and BALB/c mice, 16.7 mmol/L glucose induced a biphasic insulin release. When 1 or 5 mmol/L caffeine was included in the perifusion medium, there was a marked potentiation of the glucose-induced insulin release that was at least as responsiveness as fresh untransplanted islets. In the absence of caffeine, 3-wk-old BALB/c grafts reacted less strongly to acetylcholine than did untransplanted islets. The addition of 1 mmol/L caffeine did not enhance the potentiating effect of acetylcholine, whether in untransplanted or transplanted islets. Rather, the interaction between caffeine and acetylcholine appeared negative. We concluded that the glucose-induced insulin secretion exhibits a diminished potentiatory responsiveness to acetylcholine but not to caffeine. The displacement and denervation of transplanted islets is likely to affect either the cholinergic receptors or their mediated influence on intracellular calcium. Copyright © 1997 Elsevier Science Inc.
Effect of Fusidic Acid on Pancreatic Islet Allograft Rejection
Authors: Mendola Josá F. , Corominola Helena , Esmatjes Enric , Saenz Alex , Fernandez-Cruz Laureano , Gomis Ramon
Published: June 2017

We examined in fully mismatched rats, the survival of pancreatic islet allografts in recipients treated with either fusidic acid (FA), an antistaphyllococcal antibiotic that has been shown to possess an immunosuppressive effect in vitro and in vivo, or cyclosporin-A (CsA). Islets were isolated by collagenase digestion, separated from acinar tissue by handpicking under a dissecting microscope and transplanted into the liver by portal vein injection of streptozotocin(STZ)-induced diabetic rats. The results indicated that while a temporary immunosuppression with CsA achieved an indefinite islet allograft survival, FA administered to recipients daily was not able to prevent islet allograft rejection across a major histocompatibility barrier. We conclude that despite the fact that fusidic acid has been claimed to act as an immunosuppressant drug in vitro with effects similar to those of CsA, unlike CsA, FA given either orally or by s.c. injection was not effective to prolong islet allograft survival in vivo. Copyright © 1997 Elsevier Science Inc.
Intraretrosplenial Cortical Grafts of Fetal Cholinergic Neurons and the Restoration of Spatial Memory Function
Authors: Li Ying J. , Low Walter C.
Published: June 2017

The retrosplenial cortex (RSC) receives cholinergic afferent fibers from the medial septal nucleus and diagonal band of Broca (DBB) by way of the cingulate bundle and the fornix. Bilateral lesions of both the cingulate and fornix pathways result in a complete depletion of cholinergic input to the RSC. In the present study we have examined the effects of transplanting cholinergic neurons from fetal rat pups to the RSC of adult rats following lesions of the cingulate bundle and fornix. The animals with lesions exhibited severe spatial memory impairments with a complete loss of extrinsic cholinergic afferents to the RSC. Animals with intraretrosplenial cortical transplants exhibited significant improvements in learning and memory performance as revealed by decreased escape latencies in spatial reference memory tests, increased numbers of platform crossings in spatial navigation tests, and a higher percentage of correct choices in a spatial working memory task. These improvements appeared to be cholinergically mediated because atropine administration significantly disrupted spatial navigation performance. The survival of the transplanted cholinergic neurons and their innervation of the RSC were characterized using a monoclonal antibody to choline acetyltransferase (ChAT). The staining of graft-derived ChAT-positive fibers also revealed a pattern of innervation that mimicked that of the cholinergic input in normal animals. These results indicate that intraretrosplenial cortical transplants of cholinergic neurons can rectify spatial memory deficits produced by the loss of intrinsic cholinergic afferents from the medial septal nucleus. Copyright © 1997 Elsevier Science Inc.
Cell Proliferation in Retinal Transplants
Authors: Sharma Rajesh K. , Ehinger Berndt
Published: June 2017

The MIB-1 antibody against a nuclear protein Ki-67 was used to study the proliferation of cells in the rabbit retinal transplants. Fragmented pieces of embryonic day 15 rabbit retinas were transplanted into the subretinal space of adult rabbits and allowed to survive for different times. Fragmented donor tissue starts organizing in rosettes 1 day after transplantation. The transplanted cells continue to proliferate in the host eye and their pattern of proliferation resembles that of normal developing retina, suggesting that the factors responsible for the proliferation pattern are preserved after transplantation. The dividing cells in metaphase line up in the luminal layers of the rosettes. Certain cells become postmitotic in the regions corresponding to the inner retina first, followed by the cells in the luminal layers of rosettes. Cells in the regions between the rosettes, corresponding to the inner nuclear layer, presumably the Müller cells, proliferate significantly for the equivalent age of postnatal day 2. Few cells in these regions proliferate for at least the equivalent age of postnatal day 11 in transplants. There is a layer of nonproliferating, degenerating cells in the transplant situated close to the host retina. However, some cells in this layer, situated at the host-graft interface, proliferate. These cells proliferate for a long time possibly indicating gliosis.
Freezing Characteristics of Isolated Pig and Human Hepatocytes
Authors: Darr T.B. , Hubel A.
Published: June 2017

The cellular response of isolated hepatocytes from pigs, humans, and human hepatoblastoma cells to freezing was characterized using cryomicroscopy and analyzed using a thermodynamic model for water transport and Intracellular Ice Formation (IIF). The value for the reference permeability, Lpg, was found to be 5.8(10)-13, 1.62(10)13, and 2.7(10)-14 m/Ns for pig, human, and Hep G2/C3A cells, respectively. The activation energy, Elp, was found to be 480 kJ/mol for pig hepatocytes, 216 kJ/mol for human, and 121 kJ/mol for Hep G2/C3A cells. The average temperature at which IIF (TavgIIF) occurs was calculated to be -7.24 + 2.3°C for pig hepatocytes, -8.5 + 2.6°C for human hepatocytes, and -9.6 + 4.5°C for Hep G2/C3A cells. These results indicate that the freezing characteristics of pig and human cells are distinct and that the specific freezing characteristics need to be understood for the development of appropriate freezing protocols.
Neuropsychological Functioning following Fetal Striatal Transplantation in Huntington's Chorea: Three Case Presentations
Authors: Philpott L.M. , Kopyov O.V. , Lee A.J. , Jacques S. , Duma C.M. , Caine S. , Yang M. , Eagle K.S.
Published: June 2017

Neurotransplantation has been proposed as a potential treatment for the neurodegenerative disorder of Huntington's disease (HD), which currently has no effective therapy. While patients with Parkinson's disease have received neurotransplantation, until recently no HD patients have undergone transplantation for HD with standardized evaluations of their progress following surgery. The current report presents the cognitive changes in three patients with HD who underwent bilateral transplantation of human fetal striatal tissue. As part of the pre- and postsurgical evaluation, all three patients were administered a neuropsychological battery sensitive to the cognitive effects of HD within 2 mo prior to surgery and at 4-6 mo following transplantation. Four to 6 mo subsequent to surgery, all patients demonstrated increased scores on some measures of cognitive functioning. However, the pattern of changes was not uniform across subjects. These findings suggest that fetal striatal transplantation may improve some of the cognitive symptoms associated with HD in the three reported patients.
Colonization of Neural Allografts by Host Microglial Cells: Relationship to Graft Neovascularization
Authors: Pennell Nathan A. , Streit Wolfgang J.
Published: June 2017

In order to illuminate functional roles of microglial cells within neural allografts, we have transplanted both whole and microglial and endothelial cell-depleted E14 neural cell suspensions into the intact striatum of Sprague-Dawley rats. Following posttransplantation times of up to 30 days, the intrastrial allografts were analyzed histochemically using the Griffonia simplicifolia B4 isolectin, a marker for both microglia and blood vessels. Our results indicate that both whole and depleted suspension grafts develop identically in terms of neovascularization and microglial colonization. In both types of transplants microglial cells appeared before any blood vessels were apparent. The main phase of graft vascularization occurred between days 7 and 10 posttransplantation and neovascularization was complete by day 21, as revealed by quantitative image analysis. Microglial cells, which were present as ameboid cells during early posttransplantation times, underwent continuing cell differentiation with time that paralleled graft vascular development. By 30 days posttransplantation microglia within the grafts had assumed the fully ramified phenotype characteristic of resting adult microglia. During graft development and vascularization, microglia were often seen in close proximity to ingrowing blood vessels and vascular sprouts. In conclusion, our study has shown that microglial colonization of grafts and graft vascularization occurs independent of donor-derived microglial and endothelial cells, and suggests that the great majority of microglia and vessels within the graft are host derived. We hypothesize that the host microglia invading the allografts play an active role in promoting graft neovascularization.
Comparison of Neurotoxicity following Repeated Administration of L-Dopa, D-Dopa, and Dopamine to Embryonic Mesencephalic Dopamine Neurons in Cultures Derived from Fisher 344 and Sprague-Dawley Donors
Authors: Alexander T. , Sortwell C.E. , Sladek C.D. , Roth R. H. , Steece-Collier Kathy
Published: June 2017

Levodopa is the most efficacious and widely used symptomatic drug for Parkinson's disease (PD). There is currently, however, a great deal of interest focused on the possibility that levodopa-induced increases in dopamine (DA) turnover may increase oxidative damage derived from the breakdown of DA. Increased oxidative damage following levodopa may contribute to the progressive degeneration of remaining host nigral neurons as well as interfere with development and function of embryonic nigral neurons in neural grafting trials. There is abundant evidence that levodopa is toxic to embryonic nigral DA neurons in both cell culture and neural grafting models. These findings have prompted a number of studies on mechanisms of levodopa toxicity to identify effective means of ameliorating potential oxidative stress related to levodopa in PD. In the current study we have utilized cultures of embryonic nigral DA neurons to address the fundamental question of whether levodopa-induced toxicity is related to DA production or whether dopa itself contributes to cell death. We compared the degree of nigral DA cell death following chronic administration of: 1) levodopa (e.g.: l-dopa); 2) its less active stereoisomer d-dopa; and 3) DA. We examined the rank order of toxicity of these compounds in two species of rats, Fisher 344 (F344) and Sprague-Dawley (SD). Results indicate a toxicity profile of: DA > l-dopa > > d-dopa. In addition, although there was no difference in response of F344 and SD cultures to l-dopa, the SD cultures were significantly more susceptible to the neurotoxic effects of DA. Taken collectively, these results suggest that levodopa-induced toxicity is related primarily to DA production rather than oxidation of dopa to toxic metabolites, and that some strain related differences do exist.
Engraftment of C6-2B Cells into the Striatum of Aci Nude Rats as a Tool for Comparison of the in Vitro and in Vivo Phenotype of a Glioma Cell Line
Authors: Tornatore Carlo , Rabin Stuart , Baker-Cairns Belinda , Keir Stuart , Mocchetti Italo
Published: June 2017

The C6-2B is a well-characterized glioma cell line used extensively in the study of malignant glial biology. While the C6-2B cell line has traditionally been thought of as a homogenous cell line, the in vitro phenotype of the C6-2B cell line can vary considerably depending on the culture technique used and the stratum on which the cells are grown. Thus, we asked whether the in vitro phenotype of the C6-2B cell line was significantly different than the in vivo phenotype of the cell line once it was engrafted into the striatum of nude rats. Under culture conditions used in our laboratory, 100% of the C6 cells were found to express p75, the low-affinity nerve growth factor (NGF) receptor, and Major Histocompatability Class I (MHC Class I), while only 10-15% demonstrated vimentin reactivity. Immunohistochemistry was consistently negative for GFAP, trkA (the high-affinity receptor for NGF), CD4, CD8, and a macrophage specific marker (Ox-41). Once engrafted into the striatum of nude rats, the cells remained 100% p75 and MHC Class I positive, and again, only 15% of the cells demonstrated vimentin reactivity. The grafted cells retained this characteristic for 28 days in vivo. Although an immunoincompetent host was selected to minimize the effects an inflammatory response would have on the graft, a transient inflammatory response was detected. During the first week of engraftment, numerous MHC class II cells, some of which were macrophages, were seen infiltrating the graft. However, by 4 weeks postengraftment, no inflammatory cells were appreciated in the graft and surprisingly little reactive gliosis was seen in the penumbra of the tumor mass. Thus, the limited number of in vitro phe-notypic characteristics we examined in the C6-2B cell line remained constant once the cells were engrafted into the striatum of athymic nude rats.
Altered Differentiation of Cns Neural Progenitor Cells after Transplantation into the Injured Adult Rat Spinal Cord
Authors: Onifer Stephen M. , Cannon Allison B. , Whittemore Scott R.
Published: June 2017

Denervation of CNS neurons and peripheral organs is a consequence of traumatic SCI. Intraspinal transplantation of embryonic CNS neurons is a potential strategy for reinnervating these targets. Neural progenitor cell lines are being investigated as alternates to embryonic CNS neurons. RN33B is an immortalized neural progenitor cell line derived from embryonic rat raphé nuclei following infection with a retrovirus encoding the temperature-sensitive mutant of SV40 large T-antigen. Transplantation studies have shown that local epigenetic signals in intact or partially neuron-depleted adult rat hippocampal formation or striatum direct RN33B cell differentiation to complex multipolar morphologies resembling endogenous neurons. After transplantation into neuron-depleted regions of the hippocampal formation or striatum, RN33B cells were relatively undifferentiated or differentiated with bipolar morphologies. The present study examines RN33B cell differentiation after transplantation into normal spinal cord and under different lesion conditions. Adult rats underwent either unilateral lesion of lumbar spinal neurons by intraspinal injection of kainic acid or complete transection at the T10 spinal segment. Neonatal rats underwent either unilateral lesion of lumbar motoneurons by sciatic nerve crush or complete transection at the T10 segment. At 2 or 6-7 wk postinjury, lacZ-labeled RN33B cells were transplanted into the lumbar enlargement of injured and age-matched normal rats. At 2 wk posttransplantation, bipolar and some multipolar RN33B cells were found throughout normal rat gray matter. In contrast, only bipolar RN33B cells were seen in gray matter of kainic acid lesioned, sciatic nerve crush, or transection rats. These observations suggest that RN33B cell multipolar morphological differentiation in normal adult spinal cord is mediated by direct cell-cell interaction through surface molecules on endogenous neurons and may be suppressed by molecules released after SCI. They also indicate that the fate of immortalized neural progenitor cell lines in injured CNS must be stringently characterized.
Hypoosmotic Exposure of Canine Pancreatic Digest as a Means to Purify Islet Tissue
Authors: Lakey Jonathan R.T. , Zieger Michael A.J. , Woods Erik J. , Liu Jun , Critser John K.
Published: June 2017

The development of more effective means to separate pancreatic islets from the unwanted exocrine tissue would greatly advance the field of clinical islet allotransplantation in the treatment of insulin-dependent diabetes mellitus. Recent experiments with hamster islets have demonstrated a selective destruction of dissociated single exocrine cells when exposed to hypotonic conditions. It was the aim of this study to extend these observations to the canine model with collagenase dissociated pancreatic tissue and to evaluate the treatment's effect on islet function. Pancreases from five mongrel dogs were digested using an automated protocol of intraductal delivery of collagenase, and gentle dissociation. Duplicate samples of pancreatic digest were removed for insulin and amylase determination prior to and immediately following exposure to 50 mOsm/kg salt solution for a period of 30, 60, or 300 s before returning the digest to isoosmotic conditions. The remaining digest was cultured for a period of 48 h at 37°C before the tissue was recombined, washed, and a third sample removed for insulin and amylase. In vitro viability was then assessed using a static incubation assay with insulin content measured using a double-antibody radioimmunoassay, and amylase was determined using a colorimetric assay system. No difference in the insulin or amylase levels between the experimental groups was observed immediately following the hypotonic exposure; however, a significant decrease in the amylase content was observed following the 48-h culture period in digest that had been hypoosmotically exposed for 60 or 300 s compared with the pretreatment group (2.83 ± 0.41 IU amylase/mg pancreas vs. 1.29 ± 0.21 and 0.83 ± 0.12, mean ± SEM, p < 0.05). Insulin content was also significantly reduced in the 300-s exposure group compared with nontreated controls (3.2 ± 0.6 mU insulin/mg pancreas vs. 2.0 ± 0.2). The insulin/amylase ratio (I/A), a measure of islet and exocrine content, was 1.1 ± 0.13 following pancreas dissociation and 1.34 ± 0.21 for control tissue cultured for 48 h. The I/A ratio increased following hypoosmotic exposure to 1.50 ± 0.31 for tissue exposed for 30 s, 1.77 ± 0.19 for 60-s exposure, and 2.54 ± 0.13 for tissue exposed for 300 s (p < 0.05, vs. pretreatment group). In vitro insulin secretion was equivalent with the exception of the tissue exposed for 300 s, which had an increased basal level of insulin resulting in a significantly decreased stimulation index (3.8 ± 0.5 vs. 8.1 ± 1.2 for the purified islet control group, p < 0.05). These results suggest that a brief hypotonic exposure to pancreatic digest can alter the insulin/amylase ratio; however, there is a functional impairment on subsequent islet function after a period of in vitro tissue culture.
Highly Porous Polymer Matrices as a Three-Dimensional Culture System for Hepatocytes
Authors: Kaufmann P. M. , Heimrath S. , Kim B. S. , Moone D. J.
Published: June 2017

Hepatocyte-based therapies (e.g., hepatocyte transplantation and extracorporeal support devices) may provide alternative therapies to treat patients with liver disease, but suitable approaches to localize these cells to a given location while maintaining liver-specific gene expression must be developed. The suitability of highly porous three-dimensional sponges fabricated from poly (l-lactic acid) [PLLA] as an hepatocyte culture system was evaluated in this study. Sponges were fabricated utilizing a particulate leaching technique, and were approximately 95% porous, with an average pore diameter of 180 μm. Hepatocytes seeded into these sponges adhered and remained viable for 14 days. However, the secretion rate of albumin from these cells, an indication of liver-specific gene expression, was low (approximately 6 pg/cell/day at day 1), and decreased steadily over the 14 days of the experiment. Coating sponges with collagen, and more preferably, immobilizing cells within the PLLA sponges with a collagen gel, led to enhanced cell survival and albumin secretion at all time points. These data suggest that porous PLLA sponges may provide a novel system for long-term culture of hepatocytes, and proper design of the system may allow the liver-specific gene expression of hepatocytes transplanted in these matrices to be enhanced.
Transplantation of Xenogeneic Cells Secreting β-Endorphin for Pain Treatment: Analysis of the Ability of Components of Complement to Penetrate through Polymer Capsules
Authors: Hagihara Yasushi , Saitoh Youichi , Iwata Hiroo , Taki Takuyu , Hirano Shun-Ichiro , Arita Norio , Hayakawa Toru
Published: June 2017

The permeation of components of complement and secreted peptides through polymer capsules (PM30, K6305, and K5708) were examined. To analyze permeability by complement, the degree of hemolysis of sensitized sheep erythrocytes (EA) (1 × 109/ml) enclosed in each type of capsule was examined after 24-h incubation in culture medium containing 10% human serum. PM30 and K6305 prevented the permeation of complement well, while K5708 did not. EA suspended in alginate prevented hemolysis even in K5708. Peptide permeation through the capsules was assessed by measuring the concentration of ACTH secreted by proopiomelanocortin (POMC)-gene-transfected-Neuro2A in the culture medium on days 4, 7, 14, 21, and 28 after encapsulation. The ACTH levels in the culture medium remained high until day 28. Alginate appeared to prevent the secretion, because ACTH levels decreased in alginate-suspended cells after day 14. The PM30-K6305 double capsules containing cell lines, Neuro2A, BHK21 (hamster fibroblasts), L929 (mouse fibroblasts), and HF-SKFII (human fibroblasts) were transplanted into the cerebrospinal fluid (CSF) space of the monkeys in the lumber region. The morphological examination showed the partial survival of Neuro2A, and BHK21 and HF-SKFII, which were cells concordant with the monkeys. On the other hand, L929 cells, which were discordant with the monkeys, could not survive at all. Because these results suggest that the complement components penetrate the polymer capsules, concordant cells are preferable for xenografting with polymer capsules into the CSF space.
An Attempt to Add Biological Functions by Genetic Engineering in Order to Produce High-Performance Bioreactor Cells for Hybrid Artificial Liver: Transfection of Glutamine Synthetase into Chinese Hamster Ovary (CHO) Cell
Authors: Enosawa Shin , Suzuki Seiichi , Fujino Masayuki , Amemiya Hiroshi , Omasa Takeshi , Urayama Shigeo , Tanimura Naoko , Suga Kenichi
Published: June 2017

In the course of immortalization, hepatocyte cell lines lose their original differentiated functions, such as ammonia removal and urea formation, drug metabolism, serum protein synthesis, etc. (Enosawa et al., Cell Transplant. 5:S39-S40; 1996). With the aim of adding lost or deficient functions and producing cell lines for the bioreactor of a hybrid artificial liver, rat glutamine synthetase (GS) gene was transfected into Chinese hamster ovary (CHO) cells, because it is able to lower the ammonia level. The GS gene-inserted pSV2 plasmid was transfected into the CHO-K1 line by electroporation. Transfected CHO (GS-CHO) cells were cultured in a glutamine-free medium containing ammonia, glutamic acid, and the GS inhibitor methionine sulfoximine (MSX). The MSX concentration was increased stepwise from 25 μmol/L to 1600 μmol/L to amplify the GS gene. In several GS-CHO sublines resistant to 300-1600 μmol/L of MSX, the specific activities of GS were increased from 0.2 × 10-4 to 1.7-2.9 × 10-4 unit/106 cells. When the amplified GS-CHO cells were cultured in the ammonia-containing medium, a slow but steady decrease of the ammonia level was observed when the level was high. Finally, the prospect of genetically modulated cells for bioreactors in the hybrid artificial liver is discussed.
Microcarrier Enhanced Survival of Human and Rat Fetal Ventral Mesencephalon Cells Implanted in the Rat Striatum
Authors: Saporta Samuel , Borlongan Cesario , Moore Joann , Mejia-Millan Elizabeth , Jones Stacey L. , Bonness Patrice , Randall Timothy S. , Allen Richard C. , Freeman Thomas B. , Sanberg Paul R.
Published: June 2017

The transplantation of tissue containing dopamine-producing cells into the mammalian central nervous system is an emerging treatment for Parkinson's disease, despite relatively poor survival of implanted tissue. Recent evidence has suggested that Cytodex microcarriers enhance the survival of dopaminergic rat chromaffin cells transplanted into the rat striatum in the absence of immunosuppression. The current study was undertaken to evaluate the survival of rat and human fetal ventral mesencephalic neurons (VM) implanted alone or after attachment to microcarriers in the striatum of rats without immunosuppression. Rat fetal VM neurons demonstrated enhanced survival in the rat striatum when transplanted on microcarriers, compared to their transplantation alone during the 3-mo period examined in the present study. Transplants of human fetal VM neurons on microcarriers also survived remarkably well in the rat striatum without systemic immunosuppression. In contrast, human fetal VM cells transplanted alone into the rat striatum did not survive without systemic immunosuppression. There was no evidence of TH fiber sprouting in the vicinity of any transplant site. These data indicated that Cytodex microcarriers provide enhanced survival of both rat allograft and human xenograft fetal mesencephalic cells in the rat striatum without the necessity of systemic immunosuppression, perhaps by inducing a unique neuron–glia environment.
Follow-up Study of the Revascularization Process of Purified Rat Islet Beta-Cell Grafts
Authors: Mendola José F. , Conget Ignacio , Manzanares José María , Corominola Helena , Viñas Odette , Barcelo Juanjo , Gomis Ramon
Published: June 2017

The revascularization of islets of Langerhans transplanted in heterotopic sites like the liver by portal vein embolization or the renal subcapsular space is a major process necessary for the viability of grafted cells. This process has been extensively studied by different techniques and the results have shown that islet revascularization is an early phenomenon that takes place soon after transplantation. In this report we have analyzed by a double indirect immunofluorescence technique, the revascularization process of purified endocrine islet beta-cells transplanted in the renal subcapsular space of syngeneic rats. Lewis rats were grafted with islets cultured for 24 h, with a suspension of purified beta-cells cultured for 24 h, and with a suspension of purified beta plus nonbeta-cells cultured for 24 h. Rats were killed at different days after implantation and the kidney bearing the grafts were snap frozen and immunohistochemically stained with a rabbit anti factor VIII antiserum (which labels endothelial cells). Immunocytochemical analysis revealed that cultured islets completed revascularization by days 3-5 after transplantation, as shown by the detection of capillary endothelial cells within and surrounding the islets. Within purified endocrine beta-cell grafts, the presence of numerous endothelial cells was not observed until days 10-14, indicating that revascularization of beta-cells with host vessels is not such an early phenomenon as it takes place in whole isolated islets. Conversely, the addition of a population of endocrine nonbeta-cells to the purified islet cell grafts, partially accelerated the revascularization of pure beta-cell grafts, which showed the presence of abundant capillary endothelial cells already at day 7 after transplantation, indicating that some other unidentified factors besides the absence of endothelial cells may explain the retardation of beta-cell grafts revascularization.
Use of Polyethyleneglycol for Porcine Islet Cryopreservation
Authors: Monroy Bénédicte , Honiger Jiri , Darquy Sylviane , Reach Gérard
Published: June 2017

The aim of this work was to determine whether polyethylene glycol 20000 Da (PEG) could be used as protective agent in porcine islet cryopreservation. Cryopreservation was performed on 1-wk cultured pig islets and consisted in an overnight storage in liquid nitrogen. In a first set of experiments, we compared the in vitro function of PEG-cryopreserved islets to that of porcine islets cryopreserved under the standard procedure using dimethylsulfoxide (DMSO), by incubating the islets over 45 min in Krebs buffer containing either 2.8 or 10 mmol/L glucose. Insulin secretion of both types of islets reached a maximum at day 10 postthawing and had stimulation indices above 2 up to 3 wk after thawing. PEG-cryopreserved islets secreted more insulin than DMSO-treated islets and showed glucose-dependency insulin secretion in a 0-16.6 mmol/L glucose range. We also established that PEG-cryopreserved islets were as functional in vitro as nonfrozen tissue and that they could reverse experimental diabetes of the mouse for longer periods of time than noncryopreserved islets (p < 0.005 3 wk after transplantation) when implanted in the peritoneal cavity, being immunoprotected in a semipermeable hollow fiber. PEG can, therefore, be considered as a suitable cryoprotective compound for porcine islet storage.
Therapeutic Potential of a Polymer-Encapsulated L-Dopa and Dopamine-Producing Cell Line in Rodent and Primate Models of Parkinson's Disease
Authors: Lindner Mark D. , Emerich Dwaine F.
Published: June 2017

Encapsulation of cells within polymer membranes prior to transplantation provides a novel means of achieving continuous, site-specific delivery of therapeutic molecules to the CNS. The use of encapsulated dopamine-secreting cells that can be transplanted directly into the striatum has particular appeal for the treatment of Parkinson's disease. This article provides a brief and timely review of the progress that has been made over the past decade using encapsulated PC12 cells as a means of delivering dopamine and l-DOPA to the striatum in rodent and primate models of Parkinson's disease. The polymer membranes are well tolerated and biocompatible. Encapsulated PC12 cells survive in vivo for up to 6 mo, they release dopamine into the surrounding host striatum, and they clearly improve behavioral function in both dopamine-depleted rodents and primates. Although these results are promising, fundamental issues remain concerning the extent of dopamine diffusion from the polymer membranes and the number of devices needed for behavioral improvement, and the duration and consistency of cell viability and device output. Nevertheless, this technology appears to be a promising means of avoiding many of the practical, societal, and ethical issues that have been associated with other transplantation approaches.
Single-Cell Suspensions of Cultured Human Keratinocytes in Fibrin–Glue Reconstitute the Epidermis
Authors: Horch Raymund E. , Bannasch Holger , Kopp Jürgen , Andree Christoph , Stark G. Björn
Published: June 2017

To overcome common disadvantages of standard cultured epidermal sheet grafts (CEG) we have developed a new technique of transplanting cultured human keratinocytes suspended as single cells in a fibrin–glue matrix (Keratinocyte–fibrin–glue suspension—KFGS). In an athymic mouse model with reproducible standardized full thickness wounds this new technique was compared directly to CEG. Reepithelialization was similar in both groups, but reconstitution of the dermo-epidermal junction zone, as shown by electron microscopy and immunohistochemistry was significantly enhanced by the fibrin–glue suspension technique. The new KFGS technique is earlier available than sheet grafts, is able to transfer actively proliferative single keratinocytes, and simplifies the application.
Transplantation of Hepatocytes for Prevention of Intracranial Hypertension in Pigs with Ischemic Liver Failure
Authors: Arkadopoulos Nikolaos , Chen Steve C. , Khalili Theodore M. , Detry Olivier , Hewitt Winston R. , Lilja Helene , Kamachi Hirofumi , Petrovic Lidija , Mullon Claudy J.P. , Demetriou Achilles A. , Rozga Jacek
Published: June 2017

Intracranial hypertension leading to brain stem herniation is a major cause of death in fulminant hepatic failure (FHF). Mannitol, barbiturates, and hyperventilation have been used to treat brain swelling, but most patients are either refractory to medical management or cannot be treated because of concurrent medical problems or side effects. In this study, we examined whether allogeneic hepatocellular transplantation may prevent development of intracranial hypertension in pigs with experimentally induced liver failure. Of the two preparations tested—total hepatectomy (n = 47), and liver devascularization (n = 16)—only pigs with liver ischemia developed brain edema provided, however, that animals were maintained normothermic throughout the postoperative period. This model was then used in transplantation studies, in which six pigs received intrasplenic injection of allogeneic hepatocytes (2.5 × 109 cells/pig) and 3 days later acute liver failure was induced. In both models (anhepatic state, liver devascularization), pigs allowed to become hypothermic had significantly longer survival compared to those maintained normothermic. Normothermic pigs with liver ischemia had, at all time points studied, ICP greater than 20 mmHg. Pigs that received hepatocellular transplants had ICP below 15 mmHg until death; at the same time, cerebral perfusion pressure (CPP) in transplanted pigs was consistently higher than in controls (45 ± 11 mmHg vs. 16 ± 18 mmHg; p < 0.05). Spleens of transplanted pigs contained clusters of viable hepatocytes (hematoxylin-eosin, CAM 5.2). It was concluded that removal of the liver does not result in intracranial hypertension; hypothermia prolongs survival time in both anhepatic pigs and pigs with liver devascularization, and intrasplenic transplantation of allogeneic hepatocytes prevents development of intracranial hypertension in pigs with acute ischemic liver failure.
Evaluation of a Purified Enzyme Blend for the Recovery and Function of Canine Pancreatic Islets
Authors: Lakey Jonathan R.T. , Cavanagh Thomas J. , Zieger Michael A.J. , Wright Mary Jo
Published: June 2017

Recently developed technologies enabling the production of a reproducible, purified enzyme blend for optimal human pancreatic islet isolation has renewed interest in clinical islet transplantation. The canine model has been an ideal preclinical model for the development of islet transplantation protocols. As seen in other species, the application of crude collagenase for isolating canine islets resulted in highly variable islet yields, extensive islet fragmentation, and variable islet functionality. We compared the function of commercially available crude collagenases with that of Liberase™-CI purified enzyme blend for canine islet isolation. We also compared two manufacturing runs of Liberase-CI enzyme (lots 1 and 2) to demonstrate reproducibility of islet recovery and function. We report on the improved recovery and function of islets isolated using Liberase-CI enzyme. No difference in dog age, mean body weight, or pancreas weight were observed between the experimental groups. We observed a significantly higher postpurification recovery of islet equivalent number (IE) from pancreases processed using two lots of Liberase-CI enzyme (189 ± 20 × 103 IE, n = 4) and lot 2 (234 ± 39 × 103 IE, n = 7) than that obtained from pancreases processed with Sigma Type V (116.8 ± 27 × 103 IE, n = 5), Serva collagenase (49 ± 11.6 × 103 IE, n = 5, p < 0.05) or Boehringer–Mannheim (BM) Type P collagenase (85.4 ± 25 × 103 IE, n = 5, p < 0.05, ANOVA). No significant differences were observed in islet yield recovery from pancreases processed using the two production lots of Liberase-CI enzyme. Islet survival after 48 h in culture at 37°C was significantly higher from islets isolated using Liberase-CI enzyme (88 ± 3.7% survival) when compared to Sigma Type V (81.8 ± 3.3%), Serva (71.7 ± 2.8%), and BM Type P (77 ± 7.2%) (p < 0.05). Islet functional testing in vitro demonstrated islets isolated using crude collagenase had an increased insulin basal release and a reduced insulin stimulated response when compared with islets isolated using the two lots of Liberase-CI enzyme. The calculated stimulation index was 7.8 ± 1.7, 3.1 ± 0.6, and 4.8 ± 1.1 for Sigma Type V, Serva, and BM Type P isolated islets, respectively, compared to 15.7 ± 1.6 and 16.2 ± 1.9 for islets isolated with Liberase-CI enzyme production lots 1 and 2, respectively (p < 0.05). This evaluation demonstrates that a purified enzyme blend can significantly improve islet recovery and function. It also demonstrates the manufacturing reproducibility of Liberase-CI enzyme lots resulting in the isolation of canine islets with the same degree of efficacy. A blend of purified enzymes, specifically formulated for canine islet isolation, can consistently yield large numbers of islets that survive longer in culture and demonstrate an improved insulin response in vitro.
The Development of Intracerebral Cell-Suspension Implants is Influenced by the Grafting Medium
Authors: Watts Colin , Caldwell Maeve A. , Dunnett Stephen B.
Published: June 2017

The effect of preparing and grafting embryonic striatal and nigral tissue in four different media was evaluated in vitro and in vivo. The proportion of TH-positive and DARPP-32–positive neurons was determined after 2 days in vitro in standard culture medium following preparation in the different media. The effects were more marked for striatal neurons where DARPP-32 expression in tissue prepared in HBSS was poor compared to other media. TH expression was unaffected by the preparation medium. Striatal grafts derived from tissue prepared and grafted in HBSS were smaller, with fewer DARPP-32 cells, compared to other media. Survival of grafts in combined HBSS and DMEM was very poor. Graft volume and TH cell content was enhanced in tissue prepared in DMEM. These results suggest that preparation protocols optimized for one type of embryonic neuronal population do not necessarily transfer to other neuronal populations. © 1998 Elsevier Science Inc.
Neural Transplantation of hNT Neurons for Huntington's Disease
Authors: Hurlbert Marc S. , Gianani Rocio I. , Hutt Cynthia , Freed Curt R. , Kaddis Farida G.
Published: June 2017

Fetal striatal tissue transplants have been shown to restore motor deficits in rat and monkey models of Huntington's disease (HD). In the present study, using rats with unilateral striatal lesions, we compared fetal striatal tissue transplants to transplants of human NT (hNT) neurons. hNT neurons are terminally differentiated cells derived from the human NTera-2 cell line. In vitro, we have found that purified hNT neurons have a biochemical phenotype similar to that of human fetal striatal tissue. Both hNT neurons and fetal striatal tissue express mRNAs for glutamic acid decarboxylase, choline acetyltransferase, and the D1 and D2 dopamine receptors. Grafts of either hNT neurons or fetal striatal tissue into unilateral quinolinic acid-lesioned rat striatum improved methamphetamine-induced circling behavior. Sham controls showed no changes in methamphetamine-induced circling behavior. In the staircase test for skilled forelimb use, both transplant groups showed partial recovery in skilled use of the paw contralateral to the side of lesion, whereas the control animals showed continued deficits. These findings suggest that transplantation of hNT neurons may be an alternative to transplantation of fetal striatal tissue in the treatment of HD.
Cyclosporine A-Induced Hyperactivity in Rats: Is it Mediated by Immunosuppression, Neurotrophism, or Both?
Authors: Borlongan Cesario V. , Stahl Christine E. , Fujisaki Tomoko , Sanberg Paul R. , Watanabe Shigeru
Published: June 2017

Cyclosporine A (CsA) immunosuppressive treatment has become an adjunctive therapy in neural transplantation of dopamine-secreting cells for treatment of Parkinson's disease (PD). Recently, CsA and its analogues have been shown to promote trophic effects against neurodegenerative disorders, and therefore CsA may have direct beneficial effects on dopaminergic neurons and dopamine-mediated behaviors. The present study examined the interaction between the reported CsA-induced hyperactivity and the possible alterations in nigral tyrosine hydroxylase (TH)-immunoreactive neurons in rats with damaged blood–brain barrier. CsA was administered at a therapeutic dose (10 mg/kg/day, IP, for 9 days) used in neural transplantation protocol for PD animal models. CsA-treated animals displayed significantly higher general spontaneous locomotor activity than control animals at drug injection days 7 and 9. Histological assays at day 9 revealed that there was a significant increase in TH-immunoreactive neurons in the nigra of CsA-treated rats compared to that of the vehicle-treated rats. The nigral TH elevation was accompanied by suppressed calcium-phosphotase calcineurin activity, indicating an inhibition of host immune response. This is the first report of CsA exerting simultaneous immunosuppressive and neurotrophic effects, as well as increasing general spontaneous locomotor behavior. These results support the utility of CsA as a therapeutic agent for PD and other movement disorders.
[3H]CNQX and NMDA-Sensitive [3H]Glutamate Binding Sites and AMPA Receptor Subunit RNA Transcripts in the Striatum of Normal and Weaver Mutant Mice and Effects of Ventral Mesencephalic Grafts
Authors: Mitsacos Adamantia , Tomiyama Masahiko , Stasi Kalliope , Giompres Panayiotis , Kouvelas Elias D. , Cortés Roser , Palacios José M. , Mengod Guadalupe , Triarhou Lazaros C.
Published: June 2017

Levels of excitatory amino acid receptors were studied in the weaver mouse model of DA deficiency after unilateral intrastriatal transplantation of E12 +/+ mesencephalic cell suspensions. Graft integration was verified by turning behavior tests and from the topographical levels of the DA transporter, tagged autoradiographically with 3 nM [3H]GBR 12935 (average increase in grafted dorsal striatum compared to nongrafted side, 60%). Autoradiography of 80 nM [3H]CNQX and 100 nM NMDA-sensitive [3H]glutamate binding was carried out to visualize the topography of non-NMDA and NMDA receptors, respectively, in +/+ mice and in recipient weaver mutants 3 months after grafting. Increases of 30% or more were found for [3H]CNQX binding in the dorsal nongrafted weaver striatum compared to +/+, and a further 6–9% increase in grafted weaver compared to nongrafted side. The added increase of non-NMDA receptors in the transplanted striatum might be explained by a presence of such receptors on DA presynaptic endings of graft origin. A 20% increase in NMDA-sensitive [3H]glutamate binding was measured in the dorsal nongrafted weaver striatum compared to +/+. NMDA-sensitive [3H]glutamate binding in the transplanted side of weaver mutants tended to be slightly higher in all areas of the striatal complex compared to the nongrafted side, without reaching conventional levels of statistical significance. Using in situ hybridization histochemistry with synthetic 32P-labeled oligonucleotide probes, we investigated RNA transcripts encoding the four AMPA receptor subunits. RNA transcripts in the striatum are seen with a decreasing signal intensity in the following order: GluRB > GluRA > GluRC > GluRD. The weaver caudate-putamen shows a 12% increase in GluRA subunit mRNA compared to +/+, whereas mesencephalic neuron transplantation leads to slight increases (3%) in the levels of GluRB mRNA in the nucleus accumbens. The results are placed in the context of the important interaction between the converging glutamatergic corticostriatal and the DAergic nigrostriatal pathways in controlling the functional output of the basal ganglia in Parkinson's disease and in experimental models of DA deficiency.