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1.
Physiol Res ; 67(1): 117-126, 2018 03 16.
Article in English | MEDLINE | ID: mdl-29137485

ABSTRACT

This study aimed to investigate whether heat stress (HS) prevents a decrease in succinate dehydrogenase (SDH) activity and heat shock protein 60 (HSP60) and superoxide dismutase 2 (SOD2) contents in the extensor digitorum longus of streptozotocin (STZ)-induced diabetic rats. Twelve-week-old male Wistar rats were assigned to one of the four groups (n=6/group): control (Con), HS, diabetes mellitus (DM), and diabetes mellitus and heat stress (DM+HS). Diabetes was induced by the administration of STZ (50 mg/kg). HS was initiated 7 days after STZ treatment and performed at 42 °C for 30 min 5 times a week for 3 weeks. SDH activity was decreased in the DM and DM+HS groups. However, SDH activity was greater in the DM+HS group than in the DM group. Although HSP60 content was lower in the DM group than in the Con group, it was maintained in the DM+HS groups and was higher than that in the DM group. SOD2 content was decreased only in the DM group. These findings suggest that HS prevents the decrease in SDH activity in the skeletal muscle induced by DM. According to this mechanism, the maintenance of SOD2 and HSP60 by HS may suppress the increase in oxidative stress.


Subject(s)
Diabetes Mellitus, Experimental/metabolism , Heat-Shock Response/physiology , Muscle, Skeletal/enzymology , Succinate Dehydrogenase/metabolism , Animals , Blood Glucose/metabolism , Chaperonin 60/metabolism , Enzyme Activation/physiology , Male , Mitochondrial Proteins/metabolism , Muscle, Skeletal/metabolism , Rats , Rats, Wistar , Streptozocin , Superoxide Dismutase/metabolism
2.
Acta Physiol Hung ; 102(3): 293-300, 2015 Sep.
Article in English | MEDLINE | ID: mdl-26551745

ABSTRACT

To investigate whether heat stress attenuates skeletal muscle atrophy of the extensor digitorum longus (EDL) muscle in streptozotocin-induced diabetic rats, 12-week-old male Wistar rats were randomly assigned to four groups (n = 6 per group): control (Con), heat stress (HS), diabetes mellitus (DM), and diabetes mellitus/heat stress (DM + HS). Diabetes was induced by intraperitoneal injection of streptozotocin (50 mg/kg). Heat stress was induced in the HS and DM + HS groups by immersion of the lower half of the body in hot water at 42 °C for 30 min; it was initiated 7 days after injection of streptozotocin, and was performed once a day, five times a week for 3 weeks. The muscle fiber cross-sectional area of EDL muscles from diabetic and non-diabetic rats was determined; heat stress protein (HSP) 72 and HSP25 expression levels were also analyzed by western blotting. Diabetes-induced muscle fiber atrophy was attenuated upon heat stress treatment in diabetic rats. HSP72 and HSP25 expression was upregulated in the DM + HS group compared with the DM group. Our findings suggest that heat stress attenuates atrophy of the EDL muscle by upregulating HSP72 and HSP25 expression.


Subject(s)
Diabetes Mellitus, Experimental/complications , Heat Stress Disorders/complications , Heat-Shock Response , Hot Temperature , Muscle, Skeletal/pathology , Muscular Atrophy/prevention & control , Animals , Biomarkers/blood , Blood Glucose/metabolism , Diabetes Mellitus, Experimental/blood , Diabetes Mellitus, Experimental/pathology , HSP27 Heat-Shock Proteins/metabolism , HSP72 Heat-Shock Proteins/metabolism , Heat Stress Disorders/metabolism , Heat Stress Disorders/pathology , Male , Muscle, Skeletal/metabolism , Muscular Atrophy/etiology , Muscular Atrophy/metabolism , Muscular Atrophy/pathology , Rats, Wistar , Time Factors
3.
Acta Physiol Hung ; 101(4): 421-8, 2014 Dec.
Article in English | MEDLINE | ID: mdl-25201710

ABSTRACT

We investigated muscle atrophy, major antioxidant enzymes and lipid peroxidation in the extensor digitorum longus (EDL, predominantly fast fibers) and soleus (predominantly slow fibers) muscle of streptozotocin-diabetic rats. Female Wistar rats were divided into a control (n = 5) and streptozotocin-induced diabetic group (n = 5). Eight weeks after diabetes induction the EDL and soleus muscles were removed and catalase (CAT), glutathione peroxidase (GPX) and superoxide dismutase activity (SOD), and thiobarbituric acid reactive substances (TBARS) levels measured. The CAT activity increased in both the EDL and soleus muscles of the diabetic rats (p < 0.01), whereas the GPX and SOD activities were increased only in the EDL muscle (p < 0.01 and p < 0.05). The TBARS levels were only increased in the EDL muscle of the diabetic rats (p < 0.01). Both muscles showed significant atrophy but the EDL muscle elicited the greatest atrophy. In conclusion, it appears that adaptive responses to oxidative stress were adequate in the soleus muscle, but not in the EDL muscle, of diabetic rats. Thus fast twitch muscle fibers may be more susceptible to oxidative stress than slow twitch muscle fibers and this may contribute to muscle atrophy under diabetic conditions.


Subject(s)
Antioxidants/metabolism , Diabetes Mellitus, Experimental/enzymology , Lipid Peroxidation , Muscle, Skeletal/enzymology , Muscular Atrophy/enzymology , Streptozocin , Animals , Catalase/metabolism , Diabetes Mellitus, Experimental/chemically induced , Diabetes Mellitus, Experimental/pathology , Female , Glutathione Peroxidase/metabolism , Muscle Fibers, Fast-Twitch/enzymology , Muscle Fibers, Fast-Twitch/pathology , Muscle Fibers, Slow-Twitch/enzymology , Muscle Fibers, Slow-Twitch/pathology , Muscle, Skeletal/pathology , Muscular Atrophy/chemically induced , Muscular Atrophy/pathology , Oxidative Stress , Rats, Wistar , Superoxide Dismutase/metabolism , Thiobarbituric Acid Reactive Substances/metabolism
5.
Res Vet Sci ; 78(3): 217-24, 2005 Jun.
Article in English | MEDLINE | ID: mdl-15766940

ABSTRACT

New multidrug resistant cell lines developed from the canine B cell lymphoma cell line (GL-1) were characterized in terms of chemosensitivity to some antineoplastics and P-glycoprotein (Pgp) expression. GL-1 was continuously exposed to a culture medium containing gradually increasing levels of doxorubicin and the cells that could grow in the presence of doxorubicin were obtained. Chemosensitivity of these cells to various antineoplastics were investigated with or without verapamil, which reversed Pgp-mediated drug resistance. The expression of Pgp on the cells was also examined by Western blot analysis. As a result, three kinds of resistant cell lines, designated as GL-DOX60, 300, and 4000 were obtained. These cell lines showed stable proliferation in the medium containing 60, 300, and 4000 ng/ml, respectively. These cells were much more resistant to vincristine than doxorubicin. This resistance was strongly reversed by the presence of verapamil. On the other hand, cisplatin was effective enough in killing these derived cells. In the Western Blot analysis, some bands that reacted to the anti-human Pgp monoclonal antibodies were observed in GL-DOX4000. The cells derived from GL-1 have multidrug resistance potential mediated by canine Pgp. The cells produced in this experimental trial are considered to be useful models for various investigations on canine multidrug resistance.


Subject(s)
ATP Binding Cassette Transporter, Subfamily B, Member 1/metabolism , Antineoplastic Agents/pharmacology , Drug Resistance, Neoplasm , Lymphoma/veterinary , Animals , Cell Line, Tumor/drug effects , Cisplatin/pharmacology , Dogs , Doxorubicin/pharmacology , Drug Resistance, Multiple , Gene Expression Regulation, Neoplastic , Inhibitory Concentration 50 , Lymphoma/drug therapy , Verapamil/pharmacology , Vincristine/pharmacology
7.
Vet Comp Oncol ; 3(4): 203-10, 2005 Dec.
Article in English | MEDLINE | ID: mdl-19754775

ABSTRACT

Abstract In human and canine cancers, the inactivation of p53 protein as well as p53 gene mutation and MDM2 overexpression result in centrosome amplification that in turn contributes to chromosomal instability. To explore the usefulness of the detection of centrosome amplification as a surrogate marker of dysfunction in the p53 pathway, we systematically analysed centrosome amplification, p53 overexpression, p53 gene mutation and MDM2 overexpression in canine tumours. Centrosome amplification was detected in 16 of 51 (31%) naturally developing tumours in dogs. All the tumour specimens with aberrations in the p53 pathway, including p53 overexpression, p53 gene mutation or MDM2 overexpression, showed centrosome amplification, suggesting that the detection of centrosome amplification could serve as a preliminary surrogate marker of dysfunction in the p53 pathway.

8.
J Vet Med A Physiol Pathol Clin Med ; 49(4): 169-72, 2002 May.
Article in English | MEDLINE | ID: mdl-12069256

ABSTRACT

The purpose of this study was to evaluate the effects of the administration of an alpha2-adrenoceptor agonist alone and in combination with other derivatives on brain wave activity. In addition, the diagnostic values of the electroencephalogram (EEG) for judging the depth of the balanced anaesthesia with an alpha2-adrenoceptor agonist was evaluated. The treatments comprised 20 microg/kg medetomidine (Me-20), 80 microg/kg medetomidine (Me-80), 20 microg/kg medetomidine and 0.5 mg/kg midazolam (Me-Mi) administered intramuscularly, and 20 microg/kg medetomidine with 0.5 mg/kg midazolam and 0.1 mg/kg butorphanol (Me-Mi-Bu). The EEG was recorded continuously at pre-administration, and at 7, 10, 20, 30, 45 and 60 min after administration. The recorded data were analysed by separating the power spectrum into 1-3, 4-7, 8-13 and 14-30 Hz bands. Spectral-edge analysis was used to calculate the spectral edge frequency 90 (SEF90) and the median edge frequency (MEF). Time-related changes in power spectrum analysis showed a significant increase in the Me-80 group in the 1-3 Hz band. The power for 1-3 Hz in the Me-80 group was significantly higher than in all the other groups. In the 14-30 Hz band, there was a significant reduction of power in all groups following administration of the agents. The SEF90 frequencies were significantly reduced in all groups except for the Me-20 group after administration of the agents. The SEF90 frequencies in the Me-20, Me-Mi and Me-Mi-Bu were all significantly higher than those in the Me-80 group. However, there was no significant difference between the Me-20, Me-Mi and Me-Mi-Bu groups in any analyses. Our results demonstrated that the changes in quantitative EEG made by the Me-Mi-Bu and Me-Mi groups were similar to those made by Me-20 groups. Present results suggest that the EEG should be interpreted with caution in assessing the anaesthetic level in balanced anaesthesia in dogs.


Subject(s)
Adrenergic alpha-Agonists/pharmacology , Analgesics, Opioid/pharmacology , Brain/drug effects , Butorphanol/pharmacology , Dogs/physiology , Hypnotics and Sedatives/pharmacology , Medetomidine/pharmacology , Midazolam/pharmacology , Anesthesia/veterinary , Animals , Brain/physiology , Dose-Response Relationship, Drug , Electroencephalography/drug effects , Electroencephalography/methods , Electroencephalography/veterinary , Female , Injections, Intramuscular/veterinary , Kinetics , Male
9.
J Vet Med A Physiol Pathol Clin Med ; 49(1): 27-31, 2002 Feb.
Article in English | MEDLINE | ID: mdl-11913822

ABSTRACT

Using reverse transcription-polymerase chain reaction (RT-PCR) and semi-quantitative PCR techniques, mRNA expression for canine interferon (IFN)-gamma, interleukin (IL)-4, IL-5 and IL-10 in freshly isolated peripheral blood mononuclear cells (PBMCs) was examined in dogs with or without atopic dermatitis (AD). The expression of IFN-gamma mRNA in dogs with AD was lower than that in dogs without AD (healthy control). The expression of IL-5 mRNA was higher in dogs with AD than in control dogs, but there were no significant differences in IL-4 mRNA and IL-10 mRNA expression between the groups. The number of circulating eosinophils was higher in dogs with AD than in control dogs, although eosinophilia was found in only one dog with AD. These results suggest that there is a tendency for the PBMCs of atopic dogs to express a type 2 cytokine pattern that is similar to the pattern observed in human AD patients.


Subject(s)
Cytokines/metabolism , Dermatitis, Atopic/veterinary , Dog Diseases/immunology , Leukocytes, Mononuclear/metabolism , RNA, Messenger/blood , Animals , Case-Control Studies , Cytokines/genetics , DNA Primers , Dermatitis, Atopic/immunology , Dog Diseases/blood , Dogs , Female , Interferon-gamma/metabolism , Interleukin-10/metabolism , Interleukin-4/metabolism , Male , Polymerase Chain Reaction/veterinary , Reverse Transcriptase Polymerase Chain Reaction/veterinary
10.
Cell Transplant ; 10(7): 639-44, 2001.
Article in English | MEDLINE | ID: mdl-11714199

ABSTRACT

Pravastatin, a 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitor, is known to have suppressive effects on immune and inflammatory cells. We have previously shown in mice and dogs that this agent prevents primary nonfunction of islet iso- and autografts by reducing inflammation at the graft site. The present study was designed to further investigate whether pravastatin has a synergistic effect with cyclosporine (Cs) to prolong islet allograft survival in mice. Unpurified 3000 BALB/c newborn islets were transplanted under the renal capsule of a streptozotocin-diabetic C57BL/6 mouse. Pravastatin and Cs were administered for 10 days starting on the day of grafting (day 0). Five groups were set up based on the treatment protocol: group 1, treatment with 40 mg/kg pravastatin; group 2, 30 mg/kg Cs; group 3, 50 mg/kg Cs; group 4, 40 mg/kg pravastatin and 30 mg/kg Cs; group 5, vehicle alone. Graft survival was indicated by blood glucose levels sustained at <200 mg/dl, and graft rejection by >250 mg/dl for 2 consecutive days. Hyperglycemia persisted in six of the eight (75%) mice and grafts were rejected in 3.6 +/- 0.5 days (mean +/- SD) in group 5. In group 1, grafts were also rejected in 3.8 +/- 0.8 days, but blood glucose was transiently <200 mg/dl in three of the five mice. Despite Cs, grafts were rejected between 7 and 15 days (10.3 +/- 2.4 days) in group 2. Among six mice in group 3, one maintained euglycemia for >60 days, the other rejected the graft on day 15, and the remaining four died with functioning grafts between 9 and 13 days due to Cs toxicity. A combination of a low dose of Cs and pravastatin (group 4) prolonged graft survival for >19 days in five of the eight mice, and for 7-13 days in the remaining three mice. Histological examination of the grafts in this group showed significantly reduced local inflammation. Results indicate a synergistic effect of pravastatin and Cs on prevention of islet allograft rejection.


Subject(s)
Cyclosporine/pharmacology , Graft Survival/drug effects , Hydroxymethylglutaryl-CoA Reductase Inhibitors/pharmacology , Immunosuppressive Agents/pharmacology , Islets of Langerhans Transplantation , Pravastatin/pharmacology , Animals , Blood Glucose , Body Weight , Drug Synergism , Hyperglycemia/pathology , Kidney/pathology , Male , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Transplantation, Homologous
11.
Pancreas ; 23(1): 62-7, 2001 Jul.
Article in English | MEDLINE | ID: mdl-11451149

ABSTRACT

The digestion of pancreatic tissue with collagenase is an essential part of the islet isolation procedure. However, the process exposes islets to various types of harmful factors, including collagenase contaminants, enzymes released from the acinar cells, warm ischemia, and mechanical agitation. Nitrogen oxide production and cytokine release may also contribute to islet cell damage. Protection of islets from such damage would improve the islet yield, survival, and function. Beraprost sodium (BPS) is a prostaglandin I2 analogue, is stable in aqueous solution, and has a cytoprotective effect on various types of cells. BPS has been shown to improve the yield and function of cryopreserved and/or cultured islets. These findings prompted us to examine its cytoprotective effect on islets during the islet isolation process. Canine islets were isolated by means of a two-step digestion method and purified on Euro-Ficoll density gradient solutions (the procedure used for human islets). BPS at a concentration of 100 nM was added to the collagenase solution. After purification, the islet yield was 434,561 +/- 35.691 islet number expressed as 150 microm equivalent size (IEQ)/pancreas or 8,799 +/- 345 IEQ/g of pancreas in the BPS group and 349,987 +/- 52,887 IEQ/pancreas or 7,998 +/-1610 IEQ/g of pancreas in the control group (n = 8, each). The percent viability was 88.5 +/- 0.7% in the BPS group and 82.0 +/-0.9% in the control group (P < 0.01). Therefore, the recovery of viable islets (calculated by islet number x % viability) was 384,586 +/- 46,804 IEQ/pancreas (7,743 IEQ/g) in the BPS group and 286,989 +/- 43,367 IEQ/pancreas (6,558 IEQ/g) in the control group (P < 0.02). After culture, significantly higher numbers of islets were also recovered in the BPS group than in the control group. The islet insulin content was significantly higher in the BPS group than controls (237.8 +/- 38.5 versus 92.3 +/- 25.6 microU/IEQ; P < 0.02), although islets of both groups responded with high stimulation indices (>6). These results indicate that the addition of BPS to the collagenase solution increases the recovery of viable islets, and improves beta cell function.


Subject(s)
Cell Separation/methods , Collagenases/pharmacology , Epoprostenol/analogs & derivatives , Epoprostenol/pharmacology , Islets of Langerhans Transplantation/methods , Islets of Langerhans/cytology , Animals , Cell Count , Cell Survival/drug effects , Cells, Cultured/chemistry , Cells, Cultured/metabolism , Cells, Cultured/transplantation , Centrifugation, Density Gradient , Cryoprotective Agents/pharmacology , Dogs , Insulin/analysis , Insulin/metabolism , Insulin Secretion , Islets of Langerhans/chemistry , Islets of Langerhans/drug effects , Islets of Langerhans/metabolism , Solutions , Temperature
12.
J Vet Med Sci ; 63(12): 1331-4, 2001 Dec.
Article in English | MEDLINE | ID: mdl-11789613

ABSTRACT

A 3-month-old male Japanese cat with feline parvovirus infection, showing central and cervical nerve abnormalities, was diagnosed as hydrocephalus and syringomyelia by use of magnetic resonance imaging (MRI). The cat was maintained clinically by medical treatment even though he could not stand. The MRI scans obtained about 5 months later showed that the ventricles had increased in size and the cervical syrinx had extended into the thoracic spinal cord. Ventriculoperitoneal (VP) shunt was performed. One week after surgery, neurological conditions had improved. At the postoperative MR images, the ventricles had decreased in size and the syrinx in the cervical and thoracic spinal cord could no longer be seen. The cat was still alive and was able to walk well.


Subject(s)
Cat Diseases/diagnosis , Hydrocephalus/veterinary , Syringomyelia/veterinary , Ventriculoperitoneal Shunt/veterinary , Animals , Cat Diseases/therapy , Cats , Diuretics/therapeutic use , Furosemide/therapeutic use , Hydrocephalus/diagnosis , Hydrocephalus/therapy , Magnetic Resonance Imaging/veterinary , Male , Syringomyelia/diagnosis , Syringomyelia/therapy
13.
J Vet Med Sci ; 62(3): 305-7, 2000 Mar.
Article in English | MEDLINE | ID: mdl-10770604

ABSTRACT

Oocytes were recovered from bitch ovaries at various stages of the estrous cycle by the slicing method. The proportion of Grade A oocytes (darkly pigmented and surrounded in part, or whole, by dense layers of cumulus cells) were counted. Only Grade A oocytes were cultured in TCM199 supplemented with 5% fetal calf serum for evaluation of meiotic competence. There were no significant differences in the total number of oocytes or the proportion of Grade A oocytes that were recovered from bitches at various stages of the estrous cycle. Only 11% of the oocytes reached metaphase II (MII) at 72 hr after initiation of maturation culture. However, the proportions of oocytes reaching MII did not increase with culturing for up to 120 hr.


Subject(s)
Dogs , Oocytes/growth & development , Tissue and Organ Harvesting/veterinary , Animals , Female , Ovary/cytology , Tissue and Organ Harvesting/standards
15.
Pancreas ; 20(2): 184-90, 2000 Mar.
Article in English | MEDLINE | ID: mdl-10707935

ABSTRACT

A new approach, involving a two-step digestion process and Los Angeles preservation solution #1 (LAP-1), a cold storage solution, was developed for isolation of high-quality islets from human pancreata for transplantation. This approach markedly improves the islet yield, purity and viability, and the isolation success rate. In this method, the pancreas was digested first in warm collagenase solution for up to 20 minutes. After decanting the enzyme solution, partially digested tissue was dissociated by gentle agitation in cold LAP-1 solution without additional collagenase. The digested tissues were stored in cold LAP-1 solution until islet purification on Euro-Ficoll. Forty-six islet isolations were performed consecutively by the new method (group 1). These results were compared to those obtained earlier with 46 consecutive isolations, using our previous method that had been used before development of the new method (group 2). Our old method was a modification of Ricordi's method involving only warm collagenase digestion and the storage of digested tissues in cold Hanks balanced salt solution. All pancreata were partial, containing the body and tail. There were no significant differences in both groups with regard to the donor age, cold ischemic time, harvesting conditions, and pancreatic weight. Pancreas digestion was completed in approximately 1 hour in both groups. The isolation success rate as determined by viable islets after 2 days in culture was 93.5% (43 of 46 cases) in group 1, and 56.5% (26 of the 46) in group 2. Immediately after isolation, the new method yielded a total of 335,739 +/- 36,244 islets equivalent to 150 microm (IEQ) and 6,233 +/- 681 IEQ/g of pancreas with 83 +/- 2.5% purity, whereas the old method yielded a total of 195,587 +/- 25,242 IEQ and 3,763 +/- 5,509 IEQ/g with 69.2 +/- 4.7% purity. Isolated islets in group 1 maintained a good three-dimensional structure, displayed normal insulin release to high glucose stimulation in vitro, and restored euglycemia after transplantation into streptozotocin-diabetic athymic mice. The two-step digestion method provides a sufficient number of islets for transplantation from a single pancreas.


Subject(s)
Cell Culture Techniques/methods , Islets of Langerhans/cytology , Pancreas/cytology , Adolescent , Adult , Aged , Animals , Cells, Cultured , Child , Diabetes Mellitus, Experimental/therapy , Humans , Insulin/metabolism , Insulin Secretion , Islets of Langerhans/metabolism , Islets of Langerhans Transplantation , Mice , Middle Aged
16.
Pancreas ; 19(3): 289-96, 1999 Oct.
Article in English | MEDLINE | ID: mdl-10505760

ABSTRACT

Cryopreservation of pancreatic islets provides many advantages for clinical transplantation. Unfortunately, the freezing and thawing processes lead to a significant loss of islets. In this study, an attempt was made to increase the yield and viability of islets after cryopreservation and thawing. By using canine islets, we evaluated whether beraprost sodium (BPS), a stable prostacyclin analog, protects islets during the freeze-thaw processes and improves the recovery of frozen-thawed islets. Canine islets were frozen and thawed by the procedures used routinely for storage of human islets. In this study, we deliberately used islets of lower purity (60+/-3.6%), which is undesirable for cryopreservation. The recovery of viable islets after thawing is poorer with islets of lower purity than with highly purified islets. BPS was added to both the cryopreservation solutions containing dimethyl sulfoxide (DMSO) and the thawing solution containing sucrose. After thawing, the islet recovery (islet number after thawing divided by islet number before freezing) was 71.1+/-12.7% with 1 nM BPS, 77.8+/-5.6% with 10 nM BPS, 79.3+/-6.7% with 100 nM BPS, and 69.2+/-7.2% in control preparations without BPS. Islet viability assessed by supravital staining was 57.5+/-5.6%, 64.7+/-7.0%, 67.5+/-6.5%, and 57.7+/-4.9% with 1 nM, 10 nM, and 100 nM BPS and controls, respectively. Both islet recovery and viability were significantly better with 10 nM and 100 nM BPS than with the controls (p<0.03). After 3 days in culture, islet numbers in the 10 nM and 100 nM BPS groups were significantly higher and showed better insulin-release responses than those from the 1 nM BPS and control groups. Histologically, islet structure was well preserved in the 10 nM and 100 nM BPS groups, whereas many islets of the control group were smaller and fragmented. Electron microscopic examination revealed that 10 nM and 100 nM BPS preserved the microstructure of islet cells, and signs of apoptosis or necrosis were rare. It was concluded that BPS improved the recovery and viability of canine islets after cryopreservation and thawing. BPS would be a useful agent for improving the recovery of cryopreserved human islets for clinical transplantation.


Subject(s)
Cryopreservation , Cryoprotective Agents/pharmacology , Epoprostenol/analogs & derivatives , Islets of Langerhans/cytology , Islets of Langerhans/drug effects , Animals , Cell Count/drug effects , Cell Survival/drug effects , Culture Media, Conditioned/metabolism , Dogs , Epoprostenol/pharmacology , Insulin/biosynthesis , Islets of Langerhans/metabolism , Islets of Langerhans/ultrastructure , Microscopy, Electron
17.
Pancreas ; 17(4): 367-77, 1998 Nov.
Article in English | MEDLINE | ID: mdl-9821178

ABSTRACT

The most critical factors that affect the outcome of clinical pancreatic islet transplantation are the number and quality of donor islets available for transplantation. Toward this goal, we attempted to obtain islets that are both of better quality and higher number than are obtainable by the islet-isolation process that is now widely used. We paid special attention to two critical components of the isolation procedure: minimizing the exposure of pancreatic tissue and freed islets to warm enzyme solution, and development of a preservation solution suitable for islets during cold storage of digested pancreatic tissue-free islets. For this purpose, we developed both a two-step procedure for pancreas digestion and a new cold preservation solution, the LAP-1 solution (Los Angeles preservation solution 1). In this study, we evaluated the effect of four preservation solutions by storing digested pancreatic tissues on ice for 90 min. After the cold storage, islets were purified on three layers of Euro-Ficoll solutions in a 50-ml tube, and the islet yield, viability, and function were determined. These experiments were performed by using samples from 10 consecutive islet isolations. Results with LAP-1, original University of Wisconsin solution (oUW), and modified UW solution (mUW;UW without hydroxyethyl starch) were compared with those obtained with Hank's balanced salt solution (HBSS). The islet yield was significantly higher in the LAP-1 and mUW groups as compared with the HBSS group (p < 0.01). The islet purity was significantly better in the LAP-1, oUW, and mUW groups than the HBSS (p < 0.001). The islet viability was lowest in the HBSS group immediately after purification (vs. LAP-1, oUW, and mUW, p < 0.05) and further decreased during culture (p < 0.01). Both the number and viability of cultured islets were the highest with LAP-1 solution but without statistical significance between mUW and oUW. Electron microscopic examination showed only slight damage to cell membranes immediately after purification of islets stored in LAP-1 solution and their complete recovery within 1-2 days of culture. These islets also exhibited normal insulin responses to high glucose by static incubation and perifusion assays.


Subject(s)
Cold Temperature , Disaccharides , Islets of Langerhans/physiology , Mannitol , Tissue Preservation , Adolescent , Adult , Cell Count , Cell Separation , Cell Survival , Culture Techniques , Female , Humans , Insulin/metabolism , Insulin Secretion , Islets of Langerhans/ultrastructure , Islets of Langerhans Transplantation , Male , Microscopy, Electron , Middle Aged
18.
Transplantation ; 65(11): 1429-33, 1998 Jun 15.
Article in English | MEDLINE | ID: mdl-9645797

ABSTRACT

BACKGROUND: Nonspecific inflammatory damage in the early stages of transplantation is the major cause of primary islet graft nonfunction. Using murine isografts, we attempted to prevent this islet graft damage by treating recipients with pravastatin (Pravacol), a 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitor. Nicotinamide was also tested to determine the synergistic effect of both agents. METHODS: Unpurified newborn BALB/c islets, ranging in number from 1800 to 2500, were transplanted into the left renal subcapsular space of a syngeneic adult mouse made diabetic with streptozotocin. Recipient mice were divided into the following four groups, based on treatment protocols: treatment with 40 mg/kg pravastatin (group 1), 500 mg/kg nicotinamide (group 2), 40 mg/kg pravastatin and 500 mg/kg nicotinamide (group 3), and vehicle alone (group 4). Pravastatin and nicotinamide were administered orally every day for 14 days, starting on the day of transplantation (day 0). Nonfasting blood glucose levels, urine glucose levels, and the intravenous glucose tolerance test were used to monitor the diabetic state. The reversal of diabetes was defined by normoglycemia and negative urine glucose maintained for more than 7 days. RESULTS: After islet transplantation, levels of blood and urine glucose were significantly lower in groups 1 and 3, compared with those in group 4. K-values of an intravenous glucose tolerance test performed on day 14 were significantly higher in groups 1 and 3 than those of group 4. Reversal of diabetes had occurred in 63% of mice in group 1 and 67% in group 3, levels that were higher than those in group 2 (17%) and group 4 (0%) (P<0.02, groups 1 and 3 vs. group 4). Histological examination of grafts, biopsied on day 21, revealed well preserved islets with little sign of inflammation in groups 1 and 3, whereas grafts in groups 2 and 4 contained broken, smaller islets surrounded by severe fibrosis and mononuclear cell infiltration. CONCLUSION: Our results in mice have shown the effectiveness of pravastatin for protecting islets from nonspecific inflammatory damage. Nicotinamide did not show a synergistic effect with pravastatin at the dosage used in this study. These results indicate that pravastatin may be a useful agent for clinical islet transplantation.


Subject(s)
Hydroxymethylglutaryl-CoA Reductase Inhibitors/therapeutic use , Islets of Langerhans Transplantation/physiology , Pravastatin/therapeutic use , Animals , Animals, Newborn , Blood Glucose/analysis , Diabetes Mellitus, Experimental/drug therapy , Diabetes Mellitus, Experimental/metabolism , Diabetes Mellitus, Experimental/surgery , Glucose Tolerance Test , Glycosuria/urine , Islets of Langerhans Transplantation/pathology , Male , Mice , Mice, Inbred BALB C , Transplantation, Isogeneic
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