bluuebunny/crossref_metadata_2025 huggingface dataset:
Cytoskeletal Protein Immunoexpression in Fetal Neural Grafts: Distribution of Phosphorylated and Nonphosphorylated Neurofilament Protein and Microtubule-Associated Protein 2 (Map-2)
Authors: Rosenstein Jeffrey M. , Krum Janette M.
Published: June 2017

The present study examined the immunocytochemical expression of important cytoskeletal proteins within the neurons of an extended series of neocortical grafts and smaller group of ventral mesencephalic (nigral) grafts. Using antibodies that were directed at all three neurofilament (NF) epitopes, NF-L, NF-M, and NF-H, we attempted to determine whether these neurons would have an altered cytoskeletal profile following the stress of transplantation, because previous studies have shown such changes following ischemia or direct brain injury. We studied phosphorylated NF protein, which is found predominantly in axons, nonphosphorylated NF protein, which is found predominantly in the somata-dendritic compartment, and MAP-2, a specific microtubule marker that is localized exclusively in the somato-dendritic compartment. The results show that in all neocortical grafts examined, both phosphorylated and nonphosphorylated NF immunoexpression was significantly downregulated and appeared only in relatively few axons and somatic profiles, respectively, even though there were numerous Nissl-stained neuronal profiles in the grafts. There was no particular pattern to the immunopositive profiles. At later times occasional neuronal profiles were positive for phosphorylated NF protein, suggesting a reaction to cellular injury. In contrast to neocortical grafts, the cytoskeletal profiles of MAP-2 and phosphorylated NF protein in nigral grafts appeared very similar to age-matched control although the nonphosphorylated NF protein expression did appear somewhat lessened at 1-2 mo postoperative. Because cytoskeletal proteins play important roles in neuronal size, shape, and structural stability, they may subserve key cellular issues in neural grafting. These results show a significant loss of cytoskeletal protein expression in neocortical grafts that does not occur in nigral grafts. These results suggest that fetal neurons from different brain regions (i.e., graft source) may respond differently to the grafting procedure insofar as their cytoskeletal makeup is concerned. In addition, a potential lack of appropriate growth substrates or synaptic contacts may also produce cytoskeletal alterations. As such, the cytoskeletal protein profiles in central nervous system (CNS) grafts may be useful markers for functional performance, perhaps reflecting a degree of cellular injury.
Effects of Tectal Grafts on Sound Detection Deficits Induced by Inferior Colliculus Lesions in Hooded Rats
Authors: Zrull M.C. , Coleman J.R.
Published: June 2017

The midbrain inferior colliculus (IC), a major integrating center for auditory processing, provides a model for structural, functional, and behavioral recovery. The present study examined the role of IC in spatial sound detection, and the effects of neural transplantation in sparing of behavioral performance. Hooded rats were presented noise bursts at ambient noise levels and 15 dB above this level randomly at one of eight locations in the horizontal plane, and rats were required to suppress licking upon detecting signal presentations. Following training, rats received bilateral IC lesions, bilateral lesions followed in 1 wk by bilateral tectal grafts (embryonic Day 18), or were sham operated. During repeated testing 15 to 30 days and 40 to 45 days following surgical procedures, rats with lesions showed impaired detection task performance when compared to grafted or sham animals. Detection ratios were statistically higher for grafted rats than for rats with lesions at repeated testing times. Performance of grafted animals was not statistically separable from the sham control group; however, grafted rats never achieved preoperative detection rates nor rates as high as sham rats. Postmortem implantation of diI crystals unilaterally into grafts demonstrated fiber labeling in the ipsilateral IC and lateral lemniscus, and retrograde labeling of neurons in the remaining host IC and dorsal nucleus of lateral lemniscus. Combined with results of previous studies, the results of this study suggest that sparing of detection performance for sounds occuring at random spatial locations may be attributable to partial integration of host–graft pathways.
Bulk Cryopreservation of Isolated Islets of Langerhans
Authors: Lakey Jonathan R.T. , Warnock Garth L. , Ao Ziliang , Rajotte Ray V.
Published: June 2017

Current methods to isolate human islets of Langerhans are limited and multiple donors are required for successful reversal of longstanding Type 1 diabetes mellitus. Cryopreservation of isolated islets is an effective method of storing and pooling islets. Current cryopreservation protocols are cumbersome due to current practices of placing small aliquots of islets per individual freezer tube. In the present study, we examined the application of a blood freezer bag for the cryopreservation of isolated islets by slow cooling and rapid thawing. Freezing and thawing profiles generated using thermocouples placed inside a 500 mL Cryocyte (Baxter) blood freezer bag showed that a longer equilibration period at −7.4°C was necessary to consistently achieve nucleation and cooling profiles similar to those observed in glass tubes. When known numbers of rat islets were placed in the freezer bag and the cryoprotectant dimethyl sulfoxide (DMSO) was added in a stepwise fashion and removed using a sucrose dilution, the islet recovery compared with glass tubes was 92 ± 4.8 vs. 90 ± 2.3% (n = 4, p = ns, Mann-Whitney U-test). When purified canine islets were cryopreserved in a single freezer bag or in multiple glass tubes, the recovery was similar (78.8 ± 12.5% recovery for freezer bag vs. 82.3 ± 5.3% for glass tubes; n = 6, p = ns). In vitro function was equivalent for both groups. The stimulation index of insulin release during glucose perifusion (stimulated over basal insulin secretion) for canine islets cryopreserved in a freezer bag vs. glass tubes was 3.2 ± 1.0 and 2.3 ± 1.3, respectively (n = 6, p = ns). These values were significantly lower than the nonfrozen control islets (6.9 ± 2.4, p < 0.05). When 2000 canine islets cryopreserved in either a freezer bag, or glass tubes were transplanted into diabetic nude mice, the animals became and remained normoglycemic posttransplant. We conclude that the survival of freshly isolated canine islets cryopreserved in a single freezer bag is equivalent to the glass tube method. Bulk cryopreservation of islets in a single freezer bag will facilitate effective low temperature tissue banking to support ongoing clinical trials of islet transplantation.
A Simple Method for Cardiac Surgery in Rats
Authors: Connold A.L. , Frischknecht R. , Vrbovà G.
Published: June 2017

In our laboratory we have developed a relatively simple method for cardiac surgery in rats. The operation is carried out through a small incision in the chest wall using inexpensive equipment. This method allows for the delivery of tissue fragments and cells from a donor rat to an intact or damaged area of ventricular myocardium of a host rat, with easy subsequent localisation of the transplanted/grafted tissue. The rats recover well after the surgery and survive for long periods of time. The technique could also be used for the direct injection of chemicals or molecular probes into the heart. In our experiments we have found that embryonic rat cardiomyocytes that have been transplanted into adult host rat ventricular myocardium using this method survive and develop characteristics typical of heart muscle, thus indicating that using this technique the host heart offers a favourable environment for the transplanted embryonic heart cells.
A Potential Alternative Strategy for Myoblast Transfer Therapy: The use of Sliced Muscle Grafts
Authors: Fan Ying , Beilharz Manfred W. , Grounds Miranda D.
Published: June 2017

Excellent long-term survival (up to 1 yr) of donor skeletal muscle cells was demonstrated using a mouse Y-chromosome specific probe, following the transplantation of grafts of whole muscles from male “normal” C57B1/10Sn mice into dystrophic muscles of female host mice. After the transplantation of equivalent sliced muscle grafts there was extensive movement of the male donor cells and fusion with host myofibres. This contrasts with the extremely poor survival of isolated myoblasts after injection into the same mouse model for Duchenne muscular dystrophy. The use of sliced muscle grafts may therefore represent a potential alternative approach to myoblast transfer therapy.
Development of Fetal Sheep Pancreas after Transplantation into Athymic Mice
Authors: Tuch Bernard E. , Madrid Jocelyn C.
Published: June 2017

The capacity of the fetal sheep pancreas to grow and function when transplanted into athymic mice was examined to determine whether this source of tissue might be of potential use in reversing diabetes. For this purpose fetal sheep pancreases were obtained in the period between 50 days of gestation and fullterm (148 days). Explants (1 mm3) in organ culture secreted insulin for at least 7 days, but in steadily diminishing amounts. Acute exposure to arginine (10 mM) and theophylline (10 mM), but not glucose (20 mM), calcium chloride (10 mM), and sodium butyrate (10 mM), caused acute secretion of insulin. Explants survived for many months when grafted beneath the renal capsule of athymic mice, but their growth was less, the epithelial-like component smaller, and the percentage of endocrine cells (31 ± 5%) fewer than the case of transplanted fetal human pancreas. The β cell was the predominant endocrine cell in the ungrafted fetal sheep pancreas. In the transplanted fetal sheep pancreas this was not so, the α and PP cells being dominant—β:α:S:PP = 3:14:3:11. This pattern was unchanged when the recipient mice were hyperglycemic—β:α:δ:PP = 4:13:4:28, with no reduction of blood glucose levels being observed for up to 4 mo after transplantation. Altering the site of transplantation to the spleen or liver did not improve survival of the endocrine cells. Fetal sheep pancreatic explants when transplanted into athymic rats failed to survive. Thus, although the unusual pattern of endocrine differentiation in fetal sheep pancreas transplanted into athymic mice makes it an interesting model for further studies of fetal development, it is not of benefit in normalizing the blood glucose levels of the recipients.
Long-Term Correction of Albumin Levels in the Nagase Analbuminemic Rat: Repopulation of the Liver by Transplanted Normal Hepatocytes under a Regeneration Response
Authors: Moscioni Albert D. , Rozga Jacek , Chen Steve , Nam Arjang , Scott Henri S. , Demetriou Achilles A.
Published: June 2017

Numerous studies have reported successful transplantation of hepatocytes with demonstration of function. However, none have shown long-term correction of a liver-related metabolic defect. Male Nagase analbuminemic rats, immunosuppressed with cyclosporin-A, were transplanted with normal hepatocytes (2 × 107 cells/rat) isolated from allogeneic male Sprague–Dawley rat donors. Hepatocytes were selectively transplanted via the portal vein tributary into the posterior liver lobes of Nagase analbuminemic rats. Following 2 wk, to allow engraftment, selected transplanted rats (Group I) were reoperated and the portal venous branch supplying the anterior liver lobes was permanently ligated, resulting in their atrophy and induction of regeneration in the residual transplant-bearing lobes. Control rats consisted of: Group II—transplanted with normal hepatocytes without portal branch ligation; Group III—transplanted with analbuminemic hepatocytes with portal branch ligation; and Group IV—nontransplanted analbuminemic rats with portal branch ligation. The experimental period extended to 3 mo posttransplantation. All rats transplanted with normal hepatocytes demonstrated a significant elevation in serum albumin levels (ELISA). Group I rats had dramatic elevations in serum albumin to near normal levels (1.78 ± 0.20 g/dl), and maintained these levels until the end of the experiment Albumin levels in Group II rats reached 0.26 ± 0.07 g/dl (p < 0.001), whereas Group III and IV rats showed no changes in serum albumin levels throughout the experiment Immunohistology of liver tissue obtained from Group I rats, demonstrated large numbers (22.6 ± 7.5%) of albumin-positive hepatocytes populating the recipient liver. This is the first report of near-total and sustained correction of a genetic defect in liver function in an experimental animal model following allogeneic hepatocyte transplantation.
Pulmonary Embolism with Bone Fragments following Vertebral Body Marrow Infusion for Tolerance Induction
Authors: Fernandez Luis A. , Romaguera Rita , Viciana Ana L. , Ruiz Phillip , Tzakis Andreas G. , Ricordi Camillo
Published: June 2017

Protocols of donor bone marrow infusion for tolerance induction are receiving increasing attention in clinical trials of organ allotransplantation. We report pulmonary embolism with bone fragments following vertebral body marrow infusion in a recipient of a liver and intestinal transplant. Even though pulmonary embolism with bony microfragments has been widely described following bone marrow transplantation, the use of single, high-dose donor bone marrow infusion and/or multiple infusions currently under clinical investigation for induction of donor specific unresponsiveness, may warrant the implementation of additional steps in the vertebral body marrow processing technique to decrease or eliminate the component of bony microfragments in the final preparation.
Insulin Secretion and Glucose Tolerance after Islet Transplantation in Rats with Noninsulin-Dependent Diabetes Induced by Neonatal Streptozotocin
Authors: Tormo Maria-Angeles , Leon-Quinto Trinidad , Saulnier Catherine , Bailbe Danielle , Serradas Patricia , Portha Bernard
Published: June 2017

The present study was designed to identify in a model of noninsulin-dependent diabetes induced by neonatal streptozotocin (n0-STZ), the long-term consequences of an islet graft upon 1) glucose handling of the recipient and, 2) glucose response of the residual β cells in the recipient pancreas. We have examined, 4 and 8 wk after islet implantation under the kidney capsule of syngeneic diabetic n0-STZ rats, their tolerance to glucose administered in vivo, together with their insulin release in response to glucose in vivo (oral glucose tolerance test) as well as in vitro (perfused pancreas). The results in the islet-grafted n0-STZ rats, were compared to those obtained in nongrafted nondiabetic rats and nongrafted n0-STZ rats. Our study shows that transplanting a limited number (900) of adult islets under the kidney capsule reverses to normal, many parameters of the noninsulin-dependent diabetic state in the n0-STZ rat model: these include body weight, basal plasma glucose in both the nonfasted and postabsorptive states, and basal plasma insulin in the postabsorptive state. Furthermore, tolerance to oral glucose administration was greatly improved in the transplanted rats and it was correlated with restoration of a manifest glucose-induced insulin secretion in vivo as evaluated (ΔI) during an oral glucose tolerance test. Our data clearly show that the insulin response to glucose from the endogenous pancreas of n0-STZ diabetic rat was not really improved by long-term (8 wk) basal normoglycemia. More precisely, we were able to detect a slight but significant improvement of the early phase of insulin release in vitro in response to glucose; however, the overall insulin response remained 15 times lower than the normal one with no reapparance of the late phase of insulin release. After cessation of glucose stimulation in vivo, off-response of insulin, which is also a landmark of the impaired insulin release by the β cells of n0-STZ rats, was still detectable in the perfused pancreas of the transplanted n0-STZ rats. Finally, because the reactivity to glucose of the endogenous residual β cells was not regained, the insulin released in vivo during the oral glucose test in the graft-bearing n0-STZ rats can be attributed mainly to functioning of the grafted islets population. Copyright © 1997 Elsevier Science Inc.
Allogeneic Hepatocyte Transplantation Using FK 506
Authors: Markus P.M. , Krause P. , Fayyazi A. , Honnicke K. , Becker H.
Published: June 2017

Hepatocyte transplantation is an intriguing alternative to orthotopic liver transplantation. While engraftment of syngeneic hepatocytes can be achieved with relative ease, engraftment of allogeneic hepatocytes has been far more complicated. We used FK 506 (Tacrolimus), a novel and highly efficient immunosuppressant, which has been reported to augment liver regeneration in rats. Recipients of isolated syngeneic (LEW) and allogeneic (Wistar F.) rat hepatocytes (major histocompatibility barrier) recieved different immunosuppressive regiments with FK 506 or Cyclosporine A (CsA). Mature syngeneic hepatocytes could be retrieved up to post op day 300 with the lowest number of hepatocytes on post op day 20. Following allogeneic transplantation, no mature hepatocytes could be identified after post op day 10, though ductular like structures within the spleen were found in FK 506 but not CsA-treated animals. The epithelial cells of ductular like structures exhibit cytological features of CK-19 positive cells. Our results suggest that under CsA or FK 506 immunosuppression long-term survival of mature allogeneic hepatocytes within the spleen cannot be achieved across a major histocompatibility barrier though FK 506 allows engraftment of allogeneic donor type ductular cells. Copyright © 1997 Elsevier Science Inc.
Hybrid Muscular Tissues: Preparation of Skeletal Muscle Cell-Incorporated Collagen Gels
Authors: Okano Takahisa , Matsuda Takehisa
Published: June 2017

We prepared three different types of hybrid muscular tissues in which C2C12 cells (skeletal muscle myoblast cell line) were incorporated in type I collagen gels and then differentiated to myotubes upon culture: a disctype, a polyester mesh-reinforced sheet-type, and a tubular type. A cold mixed solution of the cells and type I collagen was poured into three different types of molds and was kept at 37°C in an incubator to form C2C12 cell-incorporated gels. A polyester mesh was incorporated into a gel to form the sheet-type tissue. The tubular hybrid tissue was prepared by pouring a mixed solution into the interstitial space of a tubular mold consisting of an outer sheath and a mandrel and subsequently culturing after removal of the outer sheath. Hybrid tissues were incubated in a growth medium (20% fetal bovine serum medium) for the first 4 days and then in a differentiation medium (2% horse serum medium) to induce formation of myotubes. Transparent fragile gels shrank with time to form opaque gels, irrespective of type, resulting in the formation of quite dense hybrid tissues. On day 14 of incubation, myoblasts fused and differentiated to form multinucleated myotubes. For a tubular type hybrid tissue, both cells and collagen fiber bundles became circumferentially oriented with incubation time. Periodic mechanical stress loading to a mesh-reinforced hybrid tissue accelerated the cellular orientation along the axis of the stretch. The potential applications for use as living tissue substitutes in damaged and diseased skeletal and cardiac muscle and as vascular grafts are discussed.
Fetal Grafting for Parkinson's Disease: Expression of Immune Markers in Two Patients with Functional Fetal Nigral Implants
Authors: Kordower Jeffrey H. , Styren Scot , Clarke Martha , Dekosky Stephen T. , Olanow C. Warren , Freeman Thomas B.
Published: June 2017

In a number of centers throughout the world, fetal nigral transplantation is being performed for the treatment of Parkinson's disease (PD). Clinical results have been inconsistent. One parameter that differs among transplant studies is the degree and manner by which patients are immunosuppressed following transplantation. Indeed, the role of the immune system following fetal grafting in humans is not well understood. Recently, two patients from our open label trial that received fetal nigral implants have come to autopsy. These patients were immunosuppressed with cyclosporin for 6 mo posttransplantation and survived for a total of 18 mo postgrafting. Robust survival of grafted dopamine-containing cells was observed in both cases. Immunostaining for HLA-DR revealed a dense collection of cells within grafts from both cases. HLA-DR staining was rarely observed within the host including non-grafted regions of the striatum. A more detailed analysis of immune markers was performed in Case 2. Numerous pan macrophages, T-cells, and B-cells were observed within graft sites located in the postcommissural putamen. In contrast, staining for these immune cells was not observed within the ungrafted anterior putamen. These findings suggest that even in healthy appearing functional nigral implants, grafts are invaded by host immune cells that could compromise their long-term viability and function. Alternatively, immune cells are known to secrete trophic factors, which may ultimately favor graft survival and function. Further work is needed to understand the role of the immune system in fetal grafting.
Addition of Lateral Ganglionic Eminence to Rat Mesencephalic Grafts Affects Fiber Outgrowth but Does not Enhance Function
Authors: Emgard-Mattson Mia , Karlsson Jenny , Nakao Naoyuki , Brundin Patrik
Published: June 2017

Addition of embryonic striatal tissue, usually as a combination of the lateral and medial ganglionic eminences, to intrastriatal mesencephalic grafts has previously been reported to enhance recovery of drug-induced rotational behavior in the host and to modify axonal fiber outgrowth from the grafted dopaminergic neurons. This study investigated the effects of adding (cografting) either lateral or medial ganglionic eminence tissue to embryonic mesencephalic grafts implanted intrastriatally, in rats with unilateral 6-hydroxydopamine lesions. The cografts did not exhibit increased survival or cell size of dopaminergic neurons when compared to transplants of mesencephalic tissue alone. Neither did recipients of cografts exhibit any enhancement of graft-induced recovery of function, when tested for drug-induced rotational behavior or forelimb function in the staircase test. However, cografts containing lateral ganglionic eminence displayed patches of dense tyrosine hydroxylase-immunoreactive fibers within the graft tissue. These patches largely coincided with patches in adjacent stained sections, which were rich in immunostaining for the striatal-specific marker dopamine- and cyclic AMP-regulated phosphoprotein-32 (DARPP-32). Such patches were not present in rats receiving cografts containing medial ganglionic eminence or mesencephalic tissue alone. Thus, it seems that the grafted dopaminergic neurons preferentially grow into the areas of the transplants containing lateral ganglionic eminence tissue. In summary, the results suggest that embryonic lateral ganglionic eminence exerts trophic effects on the outgrowth of dopaminergic axons, but does not enhance the behavioral effects of grafted dopaminergic neurons.
Successful Reversal of Diabetes by Single Donor Isologous Islet Transplantation in a Mouse Model
Authors: Leow C. K. , Gray D. W. R. , Morris P. J.
Published: June 2017

A method for isolating mouse islets which consistently gives a high yield with good purity is described. Using a bovine serum albumin gradient, the mean yield of islets per pancreas is 425 (SEM ± 15) with a consistent purity of over 90%. Single donor to single recipient of islets transplanted under the renal capsule restores normoglycemia in the diabetic recipients within 2 to 5 days of transplantation.
A Rapid and Sensitive Cellular Enzyme-Linked Immunoabsorbent Assay (CELISA) for the Detection and Quantitation of Antibodies against Cell Surface Determinants II. Optimal Reagent Concentrations and Predictive Analysis
Authors: Balkian Garo K. , Flores Kathleen M. , Llull Ramon , Ko Harry S. , Walker Kenneth W. , Black Kirby S. , Hewitt Charles W.
Published: June 2017

A cellular enzyme-linked immunosorbent assay (CELISA) was developed for the detection and quantification of antibodies elicited against allogeneic cell surface determinants. The technique uses a solid-phase cell matrix created by fixing cells with a mild formalin solution onto the bottom of a 96-well microtiter plate. A primary layer of alloantisera is first reacted against rat target cells. The secondary antibody, peroxidase conjugated antirat IgG, is then added to each well and serves as the second sandwich layer. Optimal reagent concentrations were determined by serial dilution analysis of various cell concentrations and secondary antibody dilutions. It was found that 200,000 cells per well was the optimal target cell concentration. However, 100,000 cells per well was also sufficient to run the assay with acceptable performance characteristics. Even lower cell concentrations of 10,000 and 20,000 cells/well, although not optimal, also produced acceptable results. Secondary antibody concentration with respect to the optimal cell concentration was determined to be 1:500. At 200,000 cells per well and a 1:500 secondary antibody dilution, the assay presented excellent coefficients of determination and high positive to negative ratios. The reaction was found to be very sensitive in yielding high antibody titers with low background levels and could be defined mathematically as a linear-log function. Titers of multiple unknown alloantibody samples were easily and accurately predicted in an automated manner by regression analysis form known standards. This immunoassay will be useful in studies of cell surface determinant expression and quantitation of antibodies reactive to such markers.
Evaluation of Intracerebral Grafting of Dopamine-Secreting PC12 Cells into Allogeneic and Xenogeneic Brain
Authors: Ono Takeshi , Date Isao , Imaoka Takashi , Shingo Tetsuro , Furuta Tomohisa , Asari Shoji , Ohmoto Takashi
Published: June 2017

The PC12 pheochromocytoma tumor cell line is derived from a rat adrenal medullary tumor and secretes dopamine. We have previously reported that grafted microencapsulated PC12 cells using agarose and poly(styrene sulfonic acid) survived in the xenogeneic brain without immunosuppression. To investigate whether unencapsulated PC12 cells form a tumor and how they provoke immunological reaction, PC12 cell suspension was implanted into the striatum of Sprague-Dawley rat (allogeneic graft) or guinea pig (xenogeneic graft) and histological analysis using Nissl stain and immunocytochemical analysis using antityrosine hydroxylase (TH) antibody were performed 1, 2, and 4 wk after transplantation. Host animals were not immunosuppressed. PC12 cells formed a mass 1 and 2 wk after transplantation both in allogeneic and xenogeneic brain. These grafted PC12 cells were immunoreactive to anti-TH antibody. Four weeks after transplantation, however, grafted PC12 cells in the allogeneic brain were only found within the restricted area near the site of implantation. In the xenogeneic brain, only the trace of grafted PC12 cells were found around the site of implantation 4 wk after transplantation. In both allogeneic and xenogeneic animals, a number of lymphocytes were found in and around the grafts at all period investigated. These findings indicate that PC12 cells could survive in the allogeneic or xenogeneic brain for 2 wk and were ultimately rejected by immunological reaction by 4 wk after transplantation. Implantation of encapsulated PC12 cells in the allogeneic or xenogeneic brain is considered a safe and effective method for delivering dopamine into the brain because PC12 cells will not form a tumor in the long-term even if capsules are damaged in some reason.
Prolonged Effect of Troglitazone (CS-045) on Xenograft Survival of Hybrid Artificial Pancreas
Authors: Kawakami Yoshiyuki , Inoue Kazutomo , Tun Tein , Hayashi Hiroyuki , Setoyama Hiroshi , Gu Y.J. , Cui W.X. , Imamura Masayuki , Iwata Hiroo , Ikada Yoshito
Published: June 2017

Troglitazone (CS-045), a thiazolidinedione derivative, is a new oral antidiabetic agent that enhances insulin sensitivity and improves insulin responsiveness. In this study we examined the effects of CS-045 on the survival of xenografted bioartificial pancreas. Isolated rat islets were microencapsulated with three-layer agarose microcapsules (polybrene, carboxymethyl cellulose, and an agarose-polystyrene sulfonic acid mixture). Diabetes was induced by intraperitoneal injection of streptozotocin 220 mg/kg. Recipient diabetic mice were separated into two groups. In the CS-045 treated group, the recipient mice were given feed mixed with CS-045 (0.2% w/w) starting from 1 wk before transplantation up to graft failure. The mice in the control group had feed without CS-045. Three hundred microencapsulated rat islets were xenotransplanted into the intraperitoneal cavity of each recipient mouse in both groups. One month after xenotransplantation, IVGTT was performed for all recipients. Xenotransplantation of 300 rat islets in microcapsules decreased the nonfasting blood glucose levels of both groups within 2 days. In the CS-045-treated group (n = 3), the normoglycemic period lasted for more than 1 mo without administration of immunosuppressive drugs (45 ± 4.3 days). However, in the control group (n = 4), the blood glucose levels of all recipients were already elevated on day 4. In the IVGTT study, the glucose assimilation was markedly and significantly better in the CS-045-treated group than in the control group (K = 1.7 ± 0.1 vs. 0.7 ± 0.28 respectively, p <0.01). This study demonstrates that a newly developed oral antidiabetic agent, CS-045 could favorably ameliorate the diabetic state of the recipients xenotransplanted with the bioartificial pancreas, leading to an improved glucose tolerance and longer xenograft survival.
Microchimerism and Hyporesponsiveness Induced by Intraportal Injection of Donor Spleen Cells in Rats
Authors: Ota Hirofumi , Gotoh Mitsukazu , Ohzato Hiroki , Dono Keizo , Takeda Yutaka , Umeshita Koji , Sakon Masato , Nishisho Isamu , Monden Morito
Published: June 2017

It is controversial whether or not microchimerism (MC) is responsible for the induction and maintenance of donor-specific tolerance. We have shown that intraportal injection (i.p.) of donor splenocytes induces a long-term graft survival of liver and heart in rats. In this study, we examined by polymerase chain reaction (PCR) the status of MC in the liver, spleen, and blood of rat cardiac recipients following i.p. or intravenous injection (i.v.) of donor splenocytes. Male DA (RTla) and Wistar (RTlk) rats were used as donors and recipients, respectively. Heterotopic heart transplantation was performed 10 days after i.p. or i.v. injection of 5 × 107 DA spleen cells. DA cardiac allografts were rejected with a mean survival time (MST) of 11.9 ± 1.6 (n = 10) days in nontreated recipients. Injections (i.v.) led to no significant prolongation of graft survival (MST: 11.2 ± 1.9 days, n = 6), while i.p. injection induced a significant prolongation of graft survival (MST: 18.6 ± 1.5 days, n = 5) over the control. Either i.p. or i.v. injection alone resulted in systemic MC in these organs throughout the observation time over 60 days. MC was detected in the spleen, liver, and blood of cardiac recipients 7 days after transplantation and also even after cessation of cardiac heartbeat 21 days after transplantation. This was the case with either i.p. or i.v. group, which showed MC on day 7 after transplantation and persistent MC after cessation of the heartbeat. These data suggest that the presence of MC in the liver, spleen, and the blood of transplant recipients may not be responsible for immunological unresponsiveness to donor antigens.
Survival of Nigral Grafts within the Striatum of Marmosets with 6-Ohda Lesions Depends Critically on Donor Embryo Age
Authors: Annett L. E. , Torres E. M. , Clarke D. J. , Ishida Y. , Barker R. A. , Ridley R. M. , Baker H. F. , Dunnett S. B.
Published: June 2017

The study examined the importance of embryonic donor age for the survival of nigral grafts in 6-OHDA–lesioned marmosets. The issue as to whether donor age is critical for the survival of nigral grafts in primates is controversial, because several early reports suggested that relatively old tissue could survive transplantation and produce functional benefits in monkeys, in contrast to the restrictive time dependence observed in rodents. Embryonic marmoset donors embryos of three different ages were employed: 1) E74 (Carnegie stage 18-19); 2) E83-84 (Carnegie stage 23+); 3) E92-93 (foetal period). The nigral neurons derived from the ventral mesencephalon in the two older donor age groups did not survive well when grafted to the striatum of adult marmosets with unilateral 6-OHDA lesions. Although a few tyrosine hydroxylase (TH+) neurons could be identified by immunohistochemistry at graft sites in all recipients in older donor age groups, the numbers of surviving neurons in these were small, on average typically less than 100 TH+ cells. These small grafts were not sufficient to affect amphetamine-induced rotation. In contrast, many more TH+ cells typically survived transplantation in the recipients; of graft tissue derived from the youngest donors and amphetamine-induced rotation was significantly reduced in this group alone. The time course and extent of the reduction in rotation was remarkably similar to that observed in previous marmoset nigral graft studies, confirming the utility of amphetamine-induced rotation as a sensitive and reliable indicator of nigral graft function in this species. Considering these results and other recent evidence from monkey to monkey, human to rat, and human to human graft studies, the survival of embryonic nigral tissues derived from primate donors transplanted into the striatum does appear to be critically dependent on the age of the donor tissue.
Survival and Function of Syngeneic Rat Islet Grafts Placed within the Thymus versus under the Kidney Capsule
Authors: Rayat Gina R. , Korbutt Gregory S. , Elliott John F. , Rajotte Ray V.
Published: June 2017

The role of the thymus in the ongoing acquisition of tolerance to self antigens has made it an attractive site for islet transplantation. Several studies have reported survival of rodent islet allografts in the thymus without requiring the long-term use of immunosuppressive agents; however, the degree of glucose homeostasis in the intrathymic islet transplant recipients has not been examined. We transplanted 500, 1000, or 2000 syngeneic islets into the thymus of streptozotocin-induced diabetic Wistar-Furth rats, and compared the metabolic response of these recipients with animals receiving 2000 syngeneic islets under the kidney capsule. Three of four recipients which received 2000 islets under the kidney capsule achieved normoglycemia (≤8.4 mmol/L) within 1 wk and all animals became normoglycemic within 2 wk posttransplantation. In contrast, intrathymic implantation of 2000 islets induced normoglycemia in only one of six recipients during the same time interval, and when this number was reduced to 1000 or 500 islets, none of the recipients (n = 6) normalized within 1 wk posttransplantation. Animals that received an intrathymic transplant were glucose intolerant compared to normal controls and animals with subcapsular islet transplant. Removal of the graft-bearing organs resulted in hyperglycemia in all cases, and examination of the grafts revealed the presence of numerous well-granulated insulin-containing cells in both sites. The cellular insulin content of the subcapsular grafts (67.4 ± 12.1 μg; n = 4) was significantly higher (p ≥0.05) than what was extracted from intrathymic grafts (9.5 ± 1.2 μg from 1000 islets; n = 3 and 20.0 ± 4.6 μg from 2000 islets; n = 3). We conclude that 2000 syngeneic islets implanted either in the thymus or beneath the kidney capsule can normalize hyperglycemia in streptozotocin-diabetic rats; however, normal glucose tolerance was not established in intrathymic islet recipients, suggesting that a higher number of islets may be necessary to achieve normal glucose homeostasis.
Electrostatic Endothelial Cell Transplantation within Small-Diameter (<6 MM) Vascular Prostheses: A Prototype Apparatus and Procedure
Authors: Bowlin Gary L. , Rittgers Stanley E.
Published: June 2017

This article presents a novel, clinically relevant electrostatic endothelial cell transplantation (seeding/sodding) device (U.S. & Foreign Patent Protections Pending) for small-diameter (<6 mm) vascular prostheses. The prototype apparatus was designed and built to tissue engineer 4.0 mm, I.D. GORE-TEX® (W.L. Gore & Associates, Inc.) standard wall graft segments varying in length from 4 to 12 cm. The prototype electrostatic endothelial cell transplantation apparatus is composed of an external and internal conductor, aluminum base, end supports, pillow blocks, filling apparatus, electric motor drive system, and a voltage source. The cylindrical capacitor arrangement of the device along with an electrical potential applied across the internal and external conductors creates the unique feature of this endothelial cell transplantation technique, an electric field within the cylindrical capacitor (within the graft lumen) which in turn induces a temporary positive surface charge on the graft (dielectric material) luminal surface. Multiple studies have shown that a positively charged substrate is more conducive to endothelial cell adhesion and morphological maturation (flattening) (1,2, 7,8,10,13-15). This induced positive surface charge dissipates immediately upon removal from the electrostatic endothelial cell transplantation device. Thus, after endothelial cell adhesion the graft luminal surface reverts back to its natural (nonthrombogenic) negative surface charge.
Characterization of a Fibrin Glue-Gdnf Slow-Release Preparation
Authors: Cheng Henrich , Fraidakis Matt , Blombäck Birger , Lapchak Paul , Hoffer Barry , Olson Lars
Published: June 2017

One novel method to deliver trophic factor locally in the CNS is to mix it into fibrin glue. In the present studies, [125I]-labeled GDNF-containing fibrin glue balls were used to determine binding and spread of the trophic factor. First, the binding of different concentrations of [125I]-labelèd GDNF in fibrin glue was determined in vitro. Within the six concentrations used (from 200 nM to 0.004 nM, 0 M as control), there was a strong linear correlation between the [125I]-GDNF concentration and the recovered radioactivity (r = 0.992). The mean bound radioactivity in 16 samples with 4 nM [125I]-GDNF was 71262 + 2710 CPM, and accounted for 89.8% of the mean initial count of free [125I]-GDNF (79369 + 3499 CPM). Second, [125I]-GDNF-containing glue balls were implanted into the anterior chamber of adult rats. The implanted fibrin glue balls decreased in size with time, but could still be identified on the irises 2 wk after implantation. Radioactivity was concentrated at the implantation sites in the early stages with a distribution in the surrounding iris tissue, which became separated into focal radioactive spots at the third week. Counts of radioactivity were significantly higher in the [125I]-GDNF glue ball-implanted irises than controls until 14 days after implantation. A study of the [125I] decay over time using least-squares linear regression demonstrated first-order kinetics (r = —0.98, p < 0.02) with k = 0.0091 and T 1/2 = 76 h. Finally, [125I]-GDNF–containing glue balls were implanted in the spinal cord of adult rats. Radioactivity was concentrated at the implantation sites in the early stages and was later distributed more widely in the surrounding thoracic cord. The [125I]-GDNF–containing glue degraded over time and became a porous meshwork with decreasing radioactivity at the later time points. Radioactivity in the spinal cords subjected to implantation of [125I]-GDNF–containing glue balls was higher than in controls for 14 days. Study of the [125I] decay by time with least-squares linear regression demonstrated first-order kinetics (r = -0.97, p = 0.001) with T 1/2 = 75.6 h. We conclude that the trophic factor GDNF becomes bound in the fibrin glue matrix from which it is gradually released. Our results suggest that fibrin glue is an effective substrate for keeping a trophic factor localized in situ for a finite period, protected from the circulation, surrounding aqueous humor or CSF.
The Manipulated Mesenchymal Stem Cells in Regenerated Skeletal Tissues
Authors: Nevo Zvi , Robinson Dror , Horowitz Suzanna , Hasharoni Amir , Yayon Avner
Published: June 2017

Ample experimental examples have been accumulated during the last 3 decades indicating the ability of exogenous sources of cultured cells to serve as implants accelerating cartilage regeneration in defects of articular surfaces. In some cases, the repair tissues form complete spatial reconstruction of the defect. In other cases, either the spatial reconstruction is incomplete or the quality of the reparative tissue is inadequate. A delayed pace of endochondral ossification in the deep zones of the subchondral region of the defects, or ossification above the tide mark, within the superficial cartilaginous articular regions have been noted. Therefore, even in this promising approach of biological resurfacing procedure results are not certain, and further investigative research efforts are required. In the current study, a comparison of implantations of various cultured cells of four different sources were tested in an avian system. The reparative tissue outcomes are divided into three grades: full regeneration success, partial success, and failure of regeneration according to qualitative histological parameters and quantitative observation of the gross specimen. Defects that failed to regenerate a completely filled lesion were found to contain cells carrying the preskeletal–precartilaginous characteristic marker of FGFR3. The findings based on the above parameters suggest that autogeneic, chondrocytic-enriched bone marrow derived mesenchymal cells are superior to other cell sources for articular cartilage regeneration. Grafting of defects with these cells results in a 100% success rate. Allogeneic limb bud-derived mesenchymal cells and allogeneic embryonal chondrocytes have both reached a success of 75% of completely filled defects. Allogeneic chondrocytic-enriched bone marrow-derived mesenchymal cells yielded a 31% success rate. Untreated defects completely failed to heal. In successfully healed defects no cells of the reparative tissue carry the FGFR3 marker 3 months postimplantation. In partially healed defects, FGFR3 positive staining is present in fibrous cells at the invaginated surface. These latest findings may suggest some kind of proliferation failure in such cases.
Developmental Expression of Calcium-Binding Protein-Containing Neurons in Neocortical Transplants
Authors: Rosenstein Jeffrey M. , More Newton S. , Mani Nina , Krum Janette M.
Published: June 2017

The present study examined the development of calcium binding protein-containing neurons in a timed series of fetal neocortical transplants. The immunoexpression of parvalbumin and calbindin, which are subpopulations of GABAergic neurons, have been widely studied in normal development and in disease and injury states. Because of their purported resistance to oxidative injury by their ability to buffer Ca++ influx, these neurons have been particularly studied following ischemia. Because it is likely that oxidative stress is associated with the grafting procedure, we sought to determine if these neurons displayed enhanced survival characteristics. Normally, parvalbumin and calbindin represent about 5-10% of cortical neurons. Within 2-4 wk after grafting the expression of both proteins increased markedly in that a relatively larger number of neurons (27% for parvalbumin) were immunopositive. This increase was transitory, however, and by 4 mo and beyond, confocal microscopic data showed a reduction of over 50% of parvalbumin (+) neurons and processes. Calbindin (+) processes showed a qualitative change in that they were smaller with less terminal branching. Electron microscopy confirmed a substantial reduction in parvalbumin synaptic contacts. Interestingly, in older grafts, remaining parvalbumin neurons were those that were strongly NSE (+) suggesting a link between normal metabolism and Ca++ buffering in grafted neurons. It is possible that in early grafts certain neuronal populations transiently upregulated calcium binding proteins as a defensive mechanism against Ca++ influx associated with oxidative stress. Over time, however, following physiological normalization within grafts, the calcium binding protein (+) neurons are diminished, possibly due to lack of appropriate afferent input to the interneuronal pool.
Cardiomyocyte Transplantation in a Porcine Myocardial Infarction Model
Authors: Watanabe Eiichi , Smith Duane M. , Delcarpio Joseph B. , Sun Jian , Smart Frank W. , Van Meter Clifford H. , Claycomb William C.
Published: June 2017

Transplantation of cardiomyocytes into the heart is a potential treatment for replacing damaged cardiac muscle. To investigate the feasibility and efficiency of this technique, either a cardiac-derived cell line (HL-1 cells), or normal fetal or neonatal pig cardiomyocytes were grafted into a porcine model of myocardial infarction. The myocardial infarction was created by the placement of an embolization coil in the distal portion of the left anterior descending artery in Yorkshire pigs (n = 9). Four to 5 wk after creation of an infarct, the three preparations of cardiomyocytes were grafted, at 1 × 106 cells/20 μL into normal and into the middle of the infarcted myocardium. The hearts were harvested and processed for histologic examinations 4 to 5 wk after the cell grafts. Histologic evaluation of the graft sites demonstrated that HL-1 cells and fetal pig cardiomyocytes formed stable grafts within the normal myocardium without any detrimental effect including arrhythmia. In addition, a marked increase in angiogenesis was observed both within the grafts and adjacent host myocardium. Electron microscopy studies demonstrated that fetal pig cardiomyocytes and the host myocardial cells were coupled with adherens-type junctions and gap junctions. Histologic examination of graft sites from infarct tissue failed to show the presence of grafted HL-1 cells, fetal, or neonatal pig cardiomyocytes. Cardiomyocyte transplantation may provide the potential means for cell-mediated gene therapy for introduction of therapeutic molecules into the heart.