Identification and enrichment of colony-forming cells from the adult murine pituitary.

Stem and progenitor cells have been identified in many adult tissues including bone marrow, the central nervous system, and skin. While there is direct evidence to indicate the activity of a progenitor cell population in the pituitary gland, this putative subpopulation has not yet been identified. Herein we describe the isolation and characterization of a novel clonogenic cell type in the adult murine pituitary, which we have termed Pituitary Colony-Forming Cells (PCFCs). PCFCs constitute 0.2% of pituitary cells, and generate heterogeneous colonies from single cells. PCFCs exhibit variable proliferative potential, and may exceed 11 population doublings in 14 days. Enrichment of PCFCs to 61.5-fold with 100% recovery can be obtained through the active uptake of the fluorescent dipeptide, beta-Ala-Lys-Nepsilon-AMCA. PCFCs are mostly contained within the large, agranular subpopulation of AMCA+ cells, and constitute 28% of this fraction, corresponding to 140.5-fold enrichment. Interestingly, the AMCA+ population contains rare cells that are GH+ or PRL+. GH+ cells were also identified in PCFC single cell colonies, suggesting that PCFCs have the potential to differentiate into GH+ cells. Together, these data show that the pituitary contains a rare clonogenic population which may correspond to the somatotrope/lactotrope progenitors suggested by previous experiments.

Developmentally regulated expression of the regulator of G-protein signaling gene 2 (Rgs2) in the embryonic mouse pituitary.

During the development of the anterior pituitary gland, five distinct hormone-producing cell types emerge in a spatially and temporally regulated pattern from an invagination of oral ectoderm termed Rathke's Pouch. Evidence from mouse knockout and ectopic expression studies indicates that 12.5 days post coitum (dpc) to 14.5 dpc is a critical period for the expansion of the progenitor cell pool and the determination of most hormone-secreting cell types. While signaling proteins and transcription factors have been identified as having key roles in pituitary cell differentiation, little is known about the identity and function of proteins that mediate signal transduction in progenitor cells. To identify genes that are enriched in the embryonic pituitary gland, we compared gene expression in 14.5 dpc pituitary and 14.5 dpc embryo minus pituitary tissues using the NIA 15K microarray. Analysis of the data using the R program revealed that the Regulator of G Protein Signaling 2 (Rgs2) gene was 3.9-fold more abundant in the 14.5 dpc pituitary. In situ hybridisation confirmed this finding, and showed that Rgs2 expression in midline tissues was restricted to the pituitary and discrete regions of the nervous system. Within the pituitary, Rgs2 was expressed in undifferentiated cells, and was downregulated at the completion of the hormone cell differentiation. To investigate Rgs2 function in the pituitary, we examined hormone cell differentiation in Rgs2 null neonate mice. Pituitary cell differentiation and morphology appeared normal in the Rgs2 mutant animals, suggesting that other Rgs family members with similar activities may be present in the developing pituitary.

Role of priming stresses and Hsp70 in protection from ischemia-reperfusion injury in cardiac and skeletal muscle.

Ischemia-reperfusion injury limits the survival of muscle involved in tissue trauma or transfers during microsurgical reconstruction. Priming stresses such as ischemic preconditioning or mild hyperthermia have frequently been associated with improved survival of ischemic-reperfused cardiac muscle, such protection coinciding with induction of the stress-related heat shock protein 70 (Hsp70). Little is known about the response of skeletal muscle to priming stresses. This review summarizes the current knowledge on the use of priming stresses as protective strategies against the consequences of ischemia-reperfusion in cardiac and skeletal muscle and the potential role of Hsp70.

Effects of the endothelin receptor antagonist Bosentan on ischaemia/reperfusion injury in rat skeletal muscle.

We examined the role of endothelin in ischaemia/reperfusion injury in skeletal muscle, using the endothelin receptor antagonist Bosentan. In the rat hindlimb tourniquet ischaemia model, one hindlimb was rendered ischaemic for 2 h at 36 degrees C, then blood flow was re-established for either 24 h to assess muscle survival or 1.5 h for a study of capillary perfusion. In the first set of rats, the gastrocnemius muscle was removed from the postischaemic limb and assessed for viability histochemically using the nitro blue tetrazolium stain. Tissue water content (a measure of oedema) and myeloperoxidase activity (a measure of neutrophil accumulation) were also assessed in the ischaemic muscle, the contralateral non-ischaemic muscle and the lungs. In the second set of rats, the hind limb was infused with India ink after 2-h ischaemia and 1.5-h reperfusion and the muscle was harvested, fixed and cleared. In control rats, muscle viability was 17+/-2% (S.E.M.). In rats treated with Bosentan (10 mg/kg, i.p.) 30 min before release of the tourniquet, muscle viability (48+/-7%) was significantly increased compared to the control group (P<0.01). Bosentan treatment had no significant effect on tissue water content or myeloperoxidase activity in the ischaemic muscle, the contralateral non-ischaemic muscle or the lung. Immunoreactive endothelin levels in serum increased to a peak at 90 min of reperfusion and returned to control levels by 24-h reperfusion. India ink studies demonstrated a significantly increased functional capillary density in postischaemic Bosentan-treated muscles compared with postischaemic control muscles (P<0.05). These results suggest that endothelin plays an important role in the necrosis which results from a period of ischaemia and reperfusion in skeletal muscle, by mediating a decrease in postischaemic microvascular perfusion.

Prior heat stress improves survival of ischemic-reperfused skeletal muscle in vivo.

The ability of heat stress to improve the survival of ischemic-reperfused skeletal muscle in vivo was investigated. Ischemia-reperfusion was applied using the rat hindlimb tourniquet model. The viability of ischemic-reperfused muscle (11 +/- 1%) was increased by prior mild heat stress (86 +/- 2%). To investigate whether heat shock protein 70 (Hsp 70) expression in the muscle of the heated limb was responsible for this protection, the survival of Hsp 70-expressing transduced myoblasts and myocytes was measured after exposure to mediators of ischemia-reperfusion injury. Survival was improved in Hsp 70-positive myoblasts but not in myocytes, suggesting that the mechanism of protection conferred by heat stress in vivo cannot be explained by the expression of Hsp 70 in myocytes and may involve a more complex mechanism. In conclusion, prior heat stress is effective in protecting mature skeletal muscle in vivo against necrosis after ischemia-reperfusion and has potential for use in microsurgical procedures requiring tourniquet applications.

Ischemic preconditioning: lack of delayed protection against skeletal muscle ischemia-reperfusion.

We investigated the ability of ischemic preconditioning to induce expression of heat shock protein 70 (Hsp 70) and/or to increase muscle survival after ischemia-reperfusion in the rat hind limb. Ischemic preconditioning regimens tested were; 1 x 5 min of ischemia, 4 x 5 min of ischemia interrupted by 10 min of reperfusion, 1 x 10 min of ischemia or 2 x 10 min of ischemia interrupted by 15 min of reperfusion. Western blot analysis revealed only a modest induction of Hsp 70 at 24 h after preconditioning using the latter two protocols of 1 x 10 min of ischemia or 2 x 10 min. Used at 24 h prior to prolonged ischemia, neither protocol improved muscle survival measured at 24 h after reperfusion. In conclusion, ischemic preconditioning did not produce delayed protection from ischemia-reperfusion in this model and the study suggests that ischemic preconditioning is not a useful protective strategy against skeletal muscle necrosis in the long-term.

Nitric oxide synthase-independent generation of nitric oxide in muscle ischemia--reperfusion injury.

Nitric oxide (NO) is an important molecule in many physiological or pathophysiological processes including ischemia--reperfusion injury. The enzymatic nitric oxide synthase (NOS)-dependent pathway was universally accepted as the source of NO in ischemia-reperfusion injury. However, generation of NO that is independent of NOS has also been identified in ischemia--reperfusion injury to both cardiac and skeletal muscle. This review summarizes the evidence for the generation NOS-independent NO in ischemia--reperfusion injury to cardiac and skeletal muscle.

The survival of skeletal muscle myoblasts in vitro is sensitive to a donor of nitric oxide and superoxide, SIN-1, but not to nitric oxide or peroxynitrite alone.

The survival of skeletal muscle myoblasts in culture after exposure either to a donor of NO, sodium nitroprusside (SNP), or ethanamine, 2,2'-(hydroxynitrosohydrazono)bis-(DETA NONOate), or to a donor of both NO and O(-)(2), 3-morpholinosydnonimine hydrochloride (SIN-1), was investigated. SIN-1 reduced clonogenic survival markedly but donors of NO alone did not. The injurious effect of SIN-1 was prevented by oxyhemoglobin or by uric acid but not by superoxide dismutase. The exposure of myoblasts to authentic peroxynitrite (ONOO(-)) or to DETA NONOate in the presence of an O(-)(2)-generating system did not reduce their survival. The results show that NO or ONOO(-) alone is not detrimental to myoblast survival and suggest that SIN-1 toxicity is, at least in part, mediated by H(2)O(2) in this myoblast culture system.

Nitric oxide synthase-independent generation of nitric oxide in rat skeletal muscle ischemia-reperfusion injury.

We have used electron paramagnetic resonance to investigate the time course of nitric oxide (NO) generation and its susceptibility to inhibitors of nitric oxide synthase (NOS) in ischemia-reperfusion (IR) injury to rat skeletal muscle in vivo. Significant levels of muscle nitroso-heme complexes were detected 24 h postreperfusion, but not after at 0.05, 3, and 8 h of reperfusion. The levels of muscle nitroso-heme complexes were not decreased by the NOS inhibitor N-nitro-L-arginine methyl ester as a single dose (30 mg/kg) prior to reperfusion or as multiple doses continued throughout the reperfusion (total administered, 120 mg/kg) or by the potent NOS inhibitor S-methylisothiourea (3 mg/kg). In contrast, nitroso-heme levels were reduced by the glucocorticoid dexamethasone (2.5 mg/kg). Muscle necrosis in vitro did not result in the formation of nitroso-heme complexes. The finding that reperfusion after ischemia is necessary for NO formation suggests that an inflammatory pathway is responsible for NOS-independent NO formation in IR injury to skeletal muscle.

Mild hypothermia protects against ischaemia-reperfusion injury in rabbit skeletal muscle.

In three groups of rabbits, the rectus femoris muscle was subjected to 4 hours of total ischaemia. In Group 1 (normothermia, n = 5) the core temperature was maintained within the range 36-38 degrees C for the duration of ischaemia. In Group 2 (total hypothermia, n = 5) the core temperature was allowed to fall to 31.5-33.5 degrees C. In Group 3 (muscle only hypothermia, n = 5) core temperature was maintained as in Group 1 but the muscle temperature was allowed to fall to 29.5-31.5 degrees C. After 24 hours of reperfusion the muscles were harvested and measurements made of muscle viability, oedema and myeloperoxidase content. The mean (s.e.m.) muscle viability of Group 1, 19.5 (3.8)%, was significantly less than that of both Group 2, 86.0 (2.0)%, and Group 3, 87 (4.1)%, (P < 0.001). Muscle oedema and myeloperoxidase levels were elevated in all experimental groups, but differences were not significant. These findings indicate that ischaemia-reperfusion injury in skeletal muscle in this model is highly temperature-sensitive, small reductions in muscle temperature during ischaemia providing significant protection against ischaemia-reperfusion injury.

Influence of postischemic administration of oxyradical antagonists on ischemic injury to rabbit skeletal muscle.

The aim of this study was to determine whether the administration of free radical antagonists, immediately before and during the early minutes of reperfusion, improves muscle survival 24 hr after a period of ischemia. Rabbit rectus femoris muscles were isolated, made ischemic for 3 1/2 hr and treated with either desferrioxamine (DFX), an Fe3+ chelator, superoxide dismutase and catalase (SOD & CAT), which quench superoxide and hydrogen peroxide, or allopurinol, an inhibitor of xanthine oxidase (XO). After 24 hr reperfusion, muscle viability (+/-s.e.m.), measured by the nitro blue tetrazolium (NBT) vital staining technique, was 41.6 +/- 11.3% for saline-treated ischemic controls, 30.6 +/- 7.6% for DFX-treated, 46.7 +/- 10.3% for SOD & CAT-treated, and 43.3 +/- 9.5% for allopurinol-treated muscles. None of the treated groups differed significantly from the ischemic control group. Tissue myeloperoxidase, ATP and reduced glutathione levels, and plasma lactate dehydrogenase (LDH) and aspartate transaminase (AST) levels were increased by ischemia and reperfusion in all groups, but the changes did not differ between the treatment groups. Levels of XO in the rabbit muscle were determined and found to be very low in both normal and postischemic muscle. As XO is the target enzyme of allopurinol, its absence provides a basis for the lack of effect of this agent. However, it is not clear why DFX and SOD & CAT had no protective effect.

Phosphoenolpyruvate/adenosine triphosphate enhances post-ischemic survival of skeletal muscle.

This study examined whether ischemia-reperfusion injury to skeletal muscle could be reduced by post-ischemic infusion of phosphoenolpyruvate (PEP) and adenosine triphosphate (ATP). The rectus femoris muscle of 54 rabbits was rendered ischemic for 3.5 hr. Eighteen rabbits received no further treatment. Thirty-six were infused intra-arterially at the end of ischemia, 18 with vehicle alone, and 18 with a mixture of PEP (80 mumol/kg) and ATP (2.6 mumol/kg). Six rabbits from each group were explored after 24 hr reperfusion and the muscles assessed for viability (by nitro blue tetrazolium), ATP (by luciferin-luciferase chemiluminescence), malonyldialdehyde (MDA) (thiobarbituric acid method), and water content. The remaining muscles in each group were examined histologically after either 1 hr or 4 days of reperfusion. At 24 hr the viability of the PEP/ATP infused muscles (78.9 +/- 15.4 percent) was significantly greater than that of untreated (41.4 +/- 27.3 percent) or vehicle-infused groups (34.0 +/- 32.7 percent). ATP stores were significantly higher and MDA (indicative of free radical activity) and water content significantly lower in the PEP/ATP treated group. At 24 hr and 4 days, muscles infused with PEP/ATP showed less necrosis and fewer infiltrating neutrophils than the untreated groups. Studies with isolated rabbit neutrophils showed that ATP alone significantly inhibited superoxide anion production by stimulated neutrophils. However, when combined with PEP at concentrations similar to those achieved in vivo, ATP did not significantly affect superoxide production. The findings indicate that post-ischemic infusion of PEP/ATP significantly reduces ischemia-reperfusion injury in rabbit skeletal muscle. The protective effect of PEP/ATP is more likely to be due to supplementation of intracellular ATP stores than to the inhibition of superoxide production by infiltrating neutrophils.

Platelet-activating factor (PAF) receptor antagonism by WEB 2170 improves the survival of ischaemic skeletal muscle.

The platelet-activating factor (PAF) receptor antagonist, WEB 2170, significantly improved the survival of ischaemic rabbit skeletal muscle from 42% in saline-infused muscle to 65% in WEB 2170-infused muscle at 24 hours post reperfusion. Evidence is presented which suggests that WEB 2170 inhibits neutrophil infiltration during reperfusion. In addition, tissue lipid peroxide levels and the release into blood of the enzyme creatine kinase were inhibited by local infusion of WEB 2170. In contrast, the level of oedema in muscles receiving an infusion of WEB 2170 was not different from that in saline controls. It is concluded that infusion of a PAF antagonist into ischaemic skeletal muscle at the time of reperfusion inhibits neutrophil recruitment and activation. These results provide an impetus for PAF receptor antagonism as a means of reducing reperfusion injury in limb replantation surgery.

Cool perfusion solutions for skin flaps: a new mixture of pharmacological agents which improves skin flap viability.

This study tested the hypotheses that perfusion of cooled skin flaps with established organ preservation solutions improves their viability and that this improvement can be further enhanced by pharmacological manipulation. Rabbit epigastric skin flaps were perfused with different solutions before explantation and stored at 8 degrees C for 6 days. In the first part of the experiment, flap viability was assessed 7 days after reperfusion of the flap via microvascular anastomoses. The different solutions were heparinised blood, University of Wisconsin solution, two of its modifications, EuroCollins solution and a pharmacological mixture containing phosphoenolpyruvate, desferrioxamine, nitrendipine, dextran 70 and a platelet-aggregating factor receptor antagonist (WEB 2170). In the second part, biochemical parameters of skin were measured at various reperfusion times. Adenosine triphosphate (ATP), reduced glutathione (GSH), myeloperoxidase (MPO) and tissue water were assayed at 0, 1, and 24 h after reperfusion. In addition, plasma thromboxane (TXB2) was measured at 0, 30 and 60 minutes after reperfusion. The viability of flaps perfused with the mixture (81%) was significantly higher than that of any of the other groups (39% for controls, 38% for EuroCollins, 13% for UW solution, 27% and 31% for its modifications). ATP levels after reperfusion were higher in the mixture group than in UW-perfused group. GSH levels in the mixture group were also higher than in the UW group, indicating higher level of protection against oxidative stress during reperfusion. There were no differences in MPO levels. Thromboxane levels associated with UW-perfused flaps were significantly higher than those associated with any other perfusion solution. In conclusion, perfusion of a mixture of pharmacological agents targeting specific aspects of ischaemia/reperfusion injury improved the viability of skin flaps stored in the cold for 6 days, whereas standard organ preservation solutions failed to affect significantly skin flap survival.

Autologous lymphocyte therapy for experimental canine lymphoedema: a pilot study.

Obstructive lymphoedema, an accumulation of protein-rich fluid in interstitial spaces, was created in five dogs by a combination of the irradiation of one groin and subsequent surgical ablation of any remaining lymphatics. The lymphoedema was stable for up to 2 years. The aim was to test the efficacy of intra-arterial injection of autologous lymphocytes as a therapy for lymphoedema. The hypothesis was that cytokines produced by lymphocytes mediate proteolysis by macrophage proteinases in the lymphoedematous limb to remove the excess protein and relieve the oedema. A concentrated lymphocyte-rich preparation was isolated from blood by the Ficoll-Paque method. These preparations were injected into the femoral artery four times at approximately 4 weekly intervals. Three months after the first injection of lymphocytes, lymphoedematous limbs showed a marked 69% reduction in the mean excess circumferences compared with opposite control limbs. After treatment, skin thickness and hydroxyproline content (both measures of fibrosis) as well as water content (a measure of oedema) had reduced significantly. In specimens of interstitial fluid and in skin homogenates acidic proteinase activity increased and the protein concentration decreased significantly compared with controls. It is concluded that increased proteolysis, possibly due to activated macrophages recruited to the lymphoedematous limb, may partly explain these results.

Drug mixture which improves survival of ischemic rabbit epigastric skin flaps.

The chief aim of this study was to maximize flap survival by counteracting the pathophysiological changes occurring during ischemia-reperfusion. Rabbit epigastric skin flaps given 21 hours of ischemia were infused intra-arterially with selected drugs at the start of reperfusion. Compared with control infused ischemic flaps, which had a 33% survival rate on day 7 post-ischemia, significant improvement was found with vasodilators nitrendipine (61%) and prostacyclin (65%) and the thrombolytic agent urokinase (65%); marginal improvement with the free radical scavenger desferrioxamine (53%); but no change with streptokinase (44%), heparin (21%), and ATP-MgCl2 (35%). A drug mixture comprising all of these agents except streptokinase and urokinase produced 87% survival, suggesting an additive effect. Biochemical assays on skin homogenates and blood implicated oxygen free radicals, neutrophil infiltration, and thromboxane in flap failure. These results imply that multiple factors are responsible for ischemic flap failure and that a mixture of drugs needs to be infused to counteract all of the detrimental changes.

Glycosaminoglycan composition of uninjured skin and of scar tissue in fetal, newborn and adult sheep.

Few details are available on the heterogeneity of glycosaminoglycans (GAGs) in healing fetal wound tissue. We used a sensitive assay for hexosamines to examine changes occurring in the development of normal sheep skin and of wound healing tissue in PVA sponges inserted subcutaneously at different stages of gestation. It was assumed that glucosamine was derived mainly from hyaluronan and galactosamine mainly from dermatan sulphate and chondroitin sulphate. Hexosamine-containing tissue infiltrating the sponges was deposited more rapidly in the first week than in the second week. Three days after wounding, approximately 70% of the total GAGs in wound tissue was hyaluronan. The proportion of hyaluronan then fell progressively and by the 14th day contributed 57% to the total GAGs. In uninjured skin the contribution of hyaluronan to the total GAGs fell progressively with increasing fetal maturity, the level being 70% at 75 days gestation, but only 35-40% in newborn or adult skin. At no stage of development was there a sudden change in GAG composition suggestive of a transition from regeneration to scar formation. It is concluded that hyaluronan may play an important role in the biochemical sequence leading to collagen fibrillogenesis and mature scar formation.

Collagen content of uninjured skin and scar tissue in foetal and adult sheep.

Total collagen content (measured as hydroxyproline) and Type I/Type III ratio (measured by SDS-PAGE) of normal skin and of scar tissue developing within a subcutaneously implanted polyvinyl sponge have been determined in 75, 90 and 120-day foetal lambs and adult sheep and correlated with histological appearances of the same tissues. Collagen content of normal skin is low at 75 days and rises progressively until birth when it is about half the adult level. The proportion of Type III in normal skin is highest at 75 days and falls progressively as the foetus develops. With implanted sponges the time course of changes in collagen content and I/III ratio are similar in all foetal groups and in adult sheep. Collagen content is low 3 days after implantation and rises progressively to reach a similar level in all groups by 28 days. The levels correlate closely with the amount of collagen visible in histological sections. The proportion of Type III is highest at 3 days in all groups and falls progressively as the newly formed tissue matures. The findings confirm our previous study of the healing of skin wounds that form as early as 75 days gestation foetal lambs can form scar tissue in a similar way to adult sheep.

Infusions of a novel calcitonin gene-related peptide (CGRP) derivative at the time of reperfusion to salvage ischaemic rabbit skin flaps.

Rabbit epigastric skin flaps were subjected to 21 h of ischaemia at 25 degrees C. In the first 40 min of reperfusion the flaps were infused intraarterially with either Hanks' balanced salt solution (controls), chicken CGRP or a derivative DADA-CGRP. Skin biopsies and blood specimens were taken immediately before and after 1-h reperfusion. The aim was to observe the effect of CGRP derivatives on compromised skin-flap survival and to help elucidate the critical biochemical mechanisms. It was found that chicken CGRP and DADA-CGRP produced a dose-dependent increase in blood flow, significant at and above 0.1 microgram/kg, but only the 0.1 microgram/kg DADA-CGRP infusion produced a statistically significant increase in flap survival (75.1%) as compared with controls (41.6%). CGRP infusions caused significantly more rapid restoration of tissue ATP levels and resulted in a smaller rise in blood thromboxane as compared with controls. However, CGRP caused no significant change in the tissue levels of myeloperoxidase, a measure of neutrophil infiltration, and lipid peroxidation, an indicator of free-radical activity. It is concluded that intraarterial CGRP infusions to ischaemic flaps at the time of reperfusion are indicated. However, an ideal infusion solution would also need to counteract free radicals and neutrophils which are believed to also play a major role in the inflammatory response leading to flap failure.

Coumarins: macrophage proteinase production and pinocytosis.

Coumarins, which are thought to stimulate macrophage proteinase activity, have been advocated for the treatment of high protein oedemas, such as obstructive lymphoedema. In experiments with cultured murine macrophages, coumarin and 7-hydroxycoumarin (10(-3) or 10(-4) M) had no significant effect on plasminogen-activator activity, plasminogen-independent fibrinolytic activity or pinocytosis. Although no in vitro effect on macrophage proteinase activity was found, it is possible that coumarins activate other cell types in vivo and thus effectively treat lymphoedema.