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A lentiviral construct for an enhanced green fluorescent protein (eGFP) driven by a chicken beta-actin promoter, cytomegalovirus enhancer, and intronic sequences from rabbit beta-globin (CAG) was used to produce transgenic lines of rats for evaluation of the usefulness of this approach in gene function studies. Fertilized eggs were collected from inbred Dahl S and outbred Sprague-Dawley rats, and approximately 100 pl of concentrated virus were microinjected into the perivitrelline space of one-cell embryos. Of 121 embryos injected, 60 pups (49.6%) were born. Transgenic rates averaged 22% in Dahl S and 14% in Sprague-Dawley rats. Copy number ranged from one to four in the founders, and the inheritance of the transgene in a subsequent F(1) population was 48.2%. The small number of insertion sites enabled us to derive inbred transgenic lines with a single copy of the transgene within one generation. Sequencing of each transgene insertion site revealed that they inserted as single copies with a preference for the introns of genes. The CAG promoter drove high levels of eGFP expression in brain, kidney, heart, and vasculature, making it very suitable for exploring the cardiovascular function of newly discovered genes. The pattern of eGFP expression was similar across five different F(1) transgenic lines, indicating that the expression of the transgene was independent of its chromosomal position. Thus lentiviral transgenesis provides a powerful tool for the production of transgenic inbred rats and will enhance the usefulness of this species in gene discovery and target validation studies.

The present study was designed to test the hypothesis that homocysteine (Hcys) reduces intracellular nitric oxide (NO) concentrations ([NO](i)) and stimulates superoxide (O.) production in the renal arterial endothelium, thereby resulting in endothelial dysfunction. With the use of fluorescence microscopic imaging analysis, a calcium ionophore, A-23187 (2 microM), and bradykinin (2 microM) were found to increase endothelial [NO](i) in freshly dissected lumen-opened small renal arteries loaded with 4,5-diaminofluorescein diacetate (DAF-2DA; 10 microM). Preincubation of the arteries with L-Hcys (20-40 microM) significantly attenuated the increase in endothelial [NO](i). However, L-Hcys had no effect on NO synthase activity in the renal arteries, as measured by the conversion rate of [(3)H]arginine to [(3)H]citrulline, but it concentration dependently decreased DAF-2DA-sensitive fluorescence induced by PAPA-NONOate in the solution, suggesting that L-Hcys reduces endothelial [NO](i) by its scavenging action. Because other thiol compounds such as L-cysteine and glutathione were also found to reduce [NO](i), it seems that decreased NO is not the only mechanism resulting in endothelial dysfunction or arteriosclerosis in hyperhomocysteinemia (hHcys). By analysis of intracellular O. levels using dihydroethidium trapping, we found that only L-Hcys among the thiol compounds studied markedly increased O. levels in the renal endothelium. These results indicate that L-Hcys inhibits the agonist-induced NO increase but stimulates O. production within endothelial cells. These effects of L-Hcys on [NO](i) and [O.] may contribute to endothelial injury associated with hHcys.

Substitution of chromosome 13 from Brown Norway BN/SsNHsd/Mcw (BN/Mcw) rats into the Dahl salt-sensitive SS/JrHsd/Mcw (SS/Mcw) rats resulted in substantial reduction of blood pressure salt sensitivity in this consomic rat strain designated SSBN13. In the present study, we attempted to identify genes associated with salt-sensitive hypertension by utilizing a custom, known-gene cDNA microarray to compare the mRNA expression profiles in the renal medulla (a tissue playing a pivotal role in long-term blood pressure regulation) of SS/Mcw and SSBN13 rats on either low-salt (0.4% NaCl) or high-salt (4% NaCl, 2 wk) diets. To increase the reliability of microarray data, we designed a four-way comparison experiment incorporating several levels of replication and developed a conservative yet robust data analysis method. Using this approach, from the 1,751 genes examined (representing more than 80% of all currently known rat genes), we identified 80 as being differentially expressed in at least 1 of the 4 comparisons. Substantial agreements were found between the microarray results and the results predicted on the basis of the four-way comparison as well as the results of Northern blots of 20 randomly selected genes. Analysis of the four-way comparison further indicated that approximately 75% of the 80 differentially expressed genes were likely related to salt-sensitive hypertension. Many of these genes had not previously been recognized to be important in hypertension, whereas several genes/pathways known to be involved in hypertension were confirmed. These results should provide an informative source for designing future functional studies in salt-sensitive hypertension.

We hypothesized that hindlimb suspension unloading of 8-day-old neonatal rats would disrupt the normal development of muscle fiber types and the motor innervation of the antigravity (weightbearing) soleus muscles but not extensor digitorum longus (EDL) muscles. Five rats were suspended 4.5 h and returned 1.5 h to the dam for nursing on a 24 h cycle for 9 days. To control for isolation from the dam, the remaining five littermates were removed on the same schedule but not suspended. Another litter of 10 rats housed in the same room provided a vivarium control. Fibers were typed by myofibrillar ATPase histochemistry and immunostaining for embryonic, slow, fast IIA and fast IIB isomyosins. The percentage of multiple innervation and the complexity of singly-innervated motor terminal endings were assessed in silver/cholinesterase stained sections. Unique to the soleus, unloading accelerated production of fast IIA myosin, delayed expression of slow myosin and retarded increases in standardized muscle weight and fiber size. Loss of multiple innervation was not delayed. However, fewer than normal motor nerve endings achieved complexity. Suspended rats continued unloaded hindlimb movements. These findings suggest that motor neurons resolve multiple innervation through nerve impulse activity, whereas the postsynaptic element (muscle fiber) controls endplate size, which regulates motor terminal arborization. Unexpectedly, in the EDL of unloaded rats, transition from embryonic to fast myosin expression was retarded. Suspension-related foot drop, which stretches and chronically loads EDL, may have prevented fast fiber differentiation. These results demonstrate that neuromuscular development of both weightbearing and non-weightbearing muscles in rats is dependent upon and modulated by hindlimb loading.

Spacelab Life Sciences-1 and -2 provided skeletal muscles from rats dissected in flight for the first time and 2 h to 14 days postflight. The muscles permitted the distinguishing of primary adaptations to microgravity from secondary reloading-induced alterations. In microgravity, rats adopted bipedal forelimb locomotion with the hindlimbs relegated to grasping activities. On landing day, body posture was abnormally low and walking was stilted at a rate one-third of normal. The adductor longus (AL) and soleus muscles exhibited decreased myofiber areas that did not recover 14 days postflight. Doubling of the nonmyofiber area indicated interstitial edema in AL muscles 2.3 h postflight. Solei did not manifest edema postflight, and neither muscle showed edema in flight. Sarcomere eccentric contraction-like lesions were detected in 2.6% of AL myofibers 4.5 h postflight; lesions were absent earlier postflight and in flight. At 9 days postflight, these lesions were repaired but regenerating AL myofibers were present, which suggests that myofiber necrosis occurred 1-2 days postflight. These studies demonstrate that muscle atrophy occurs in microgravity, whereas interstitial edema and sarcomere lesions are postflight phenomena.

Hindlimb suspension unloading (HSU) and spaceflight microgravity induce atrophy of the slow adductor longus muscle fibers which, following reloading, exhibit eccentric contraction (EC)-like lesions (abnormal widening of sarcomeres with A band disruption and excessively wavy, extracted Z lines). These lesions are similar morphologically to those produced in normal muscles after strenuous eccentric exercise. It appears that atrophic muscles exhibit increased susceptibility to eccentric damage because lesions are produced during nonstressful voluntary movements upon return to weightbearing. The EC-like lesions are absent in the unweighted conditions, but appear in HSU rats 15-60 minutes after reloading and in space-flown rates about 4 hrs after landing. By 12 hours, many EC-like lesioned sarcomeres are fully covered by longitudinal patches of Z line-like material which increases in density by 48 hours, producing the so-called "Z line streaming" morphology. In this case, Z line streaming is indicative of rapid repair of damaged sarcomeres rather than the onset of sarcomere breakdown. Immunoelectron microscopy is necessary to determine the composition of this dense material. By 9 days of reloading at 1 gravity, sarcomeres have regained normal structure, except for very rare persistence of faint Z patches. The morphological data indicate that Z patches serve at least two functions: 1) to permit contractile force to be transmitted across the damaged sarcomeres and 2) to provide a scaffold upon which sarcomeres are reconstructed in an active functional muscle.

Spaceflight (flight) and tail suspension-hindlimb unloading (unloaded) produced significant decreases in fiber cross-sectional areas of the adductor longus (AL), a slow-twitch antigravity muscle. However, the mean wet weight of the flight AL muscles was near normal, whereas that of the suspension unloaded AL muscles was significantly reduced. Interstitial edema within the flight AL, but not in the unloaded AL, appeared to account for this apparent disagreement. In both experimental conditions, the slow-twitch oxidative fibers atrophied more than the fast-twitch oxidative-glycolytic and fast-twitch glycolytic fibers. Immunostaining showed that slow-twitch oxidative fibers expressed fast myosin, producing hybrid fibers containing slow and fast myosin isoforms. Two-dimensional gel electrophoresis of flight AL muscles revealed increased content of fast myosin light chains and decreased amounts of slow myosin light chains and fatty acid-binding protein. In the flight AL, absolute mitochondrial content decreased, but the relatively greater breakdown of myofibrillar proteins maintained mitochondrial concentration near normal in the central intermyofibrillar regions of fibers. Subsarcolemmal mitochondria were preferentially lost and reduced below normal concentration. Elevated fiber immunostaining for ubiquitin conjugates was suggestive of ubiquitin-mediated breakdown of myofibrillar proteins. On return to weight bearing for 8-11 h, the weakened atrophic muscles exhibited eccentric contraction-like lesions (hyperextension of sarcomeres with A-band filaments pulled apart and fragmented), tearing of the supporting connective tissue, and thrombosis of the microcirculation. Segmental necrosis of muscle fibers, denervation of neuromuscular junctions, and extravasation of red blood cells were minimal. Lymphocyte antibody markers did not indicate a significant immune reaction. The flight AL exhibited threefold more eccentric-like lesions than the unloaded AL; the high reentry G forces experienced by the flight animals, but not the unloaded group, possibly accounted for this difference. Muscle atrophy appears to increase the susceptibility to form eccentric contraction-like lesions after reloading; this may reflect weakening of the myofibrils and extracellular matrix. Microcirculation was also compromised by spaceflight, such that there was increased formation of thrombi in the post-capillary venules and capillaries. This blockage led to edema by 8-11 h after resumption of weight bearing by the COSMOS 2044 rats. The present findings indicate that defective microcirculation most likely accounted for the extensive tissue necrosis and microhemorrhages observed for COSMOS 1887 rats killed 2 days after landing.

Soleus muscle atrophy was induced by hindlimb unloading of male Sprague-Dawley rats (305 +/- 15 g) for 4, 7, and 10-14 days. Controls (291 +/- 14 g) were housed in vivarium cages. Soleus electromyogram (EMG) activity was recorded before and during tail suspension. Unloading caused progressive reduction in the muscle-to-body weight ratio. After 14 days, type I and IIa fibers decreased in area 63 and 47%, respectively. Subsarcolemmal mitochondria and myofibrils were degraded more rapidly than intermyofibrillar mitochondria and the cell membrane. After 10 days, 3% of the fibers exhibited segmental necrosis; affected fibers were all high-oxidative type IIa fibers. This suggested ischemic injury. By 13 days, 30% of the fibers possessed central corelike lesions involving primarily type I fibers. Video monitoring revealed abnormal plantar flexion of the hindfeet by 4 days; this posture shortened the soleus working range. Corelike lesions indicated adaptation to the shortened length. No morphological signs of denervation were detected. EMG activity shifted from tonic to phasic, and aggregate activity was 13% of normal after 7 days. These findings indicate that the atrophy and pathological changes result from unloaded contractions, reduced use, compromised blood flow, and shortened working length.

Histochemical and ultrastructural analyses were performed postflight on hind limb skeletal muscles of rats orbited for 12.5 days aboard the unmanned Cosmos 1887 biosatellite and returned to Earth 2 days before sacrifice. The antigravity adductor longus (AL), soleus, and plantaris muscles atrophied more than the non-weight-bearing extensor digitorum longus, and slow muscle fibers were more atrophic than fast fibers. Muscle fiber segmental necrosis occurred selectively in the AL and soleus muscles; primarily, macrophages and neutrophils infiltrated and phagocytosed cellular debris. Granule-rich mast cells were diminished in flight AL muscles compared with controls, indicating the mast cell secretion contributed to interstitial tissue edema. Increased ubiquitination of disrupted myofibrils implicated ubiquitin in myofilament degradation. Mitochondrial content and succinic dehydrogenase activity were normal, except for subsarcolemmal decreases. Myofibrillar ATPase activity of flight AL muscle fibers shifted toward the fast type. Absence of capillaries and extravasation of red blood cells indicated failed microcirculation. Muscle fiber regeneration from activated satellite cells was detected. About 17% of the flight AL end plates exhibited total or partial denervation. Thus, skeletal muscle weakness associated with spaceflight can result from muscle fiber atrophy and segmental necrosis, partial motor denervation, and disruption of the microcirculation.

Contraction damage occurring when longitudinal frozen sections of fresh unfixed muscles are thawed on microscope slides has limited histological examination of this tissue mainly to cross sections. Longitudinally oriented sections are advantageous for investigating properties that vary along the length of the muscle fibers. A fume fixation technique has been developed for preventing contraction of thick longitudinal frozen sections. The technique is compatible with histochemical staining of enzymes.

The pattern of nerves, ganglia, and fine nerve processes in the adult rabbit sinoatrial node, identified by microelectrode recording, was defined by staining histochemically for cholinesterase followed by silver impregnation. A generalized repeatable pattern of innervation was recognized, including 1) a large ganglionic complex inferior to the sinoatrial node; 2) two or three moderately large nerves traversing the sinoatrial node parallel to the crista terminalis; 3) nerves entering the region from the atrial septum, the superior vena cava, and the inferior vena cava; and 4) a fine network of nerve processes, particularly extensive in the morphologically dense small-cell part of the sinoatrial node. When the site of initial depolarization in the node was located and marked by a broken-off electrode tip, it was found, after cholinesterase staining, to be characterized by a cluster of cells enclosed in a nest or basket of fine nerves. Similar nested cell clusters were observed elsewhere in the sinoatrial node in this same preparation and in other hearts. A complex interweaving of atrial muscle fibers was observed medial and inferomedial to the sinoatrial node, which may form the anatomical basis for the lack of conduction through this region. The morphological pattern of nerves, ganglia, and myocardial cells described in this study emphasizes the complexity of innervation of the sinoatrial node, including its intrinsic neural elements. Cholinesterase/silver staining can be useful in the definition and comparison of electrophysiologically identified sites within the sinoatrial node.

Prolonged exposure of humans to hypogravity causes weakening of their skeletal muscles. This problem was studied in rats exposed to hypogravity for 7 days aboard Spacelab 3. Hindlimb muscles were harvested 12-16 hours postflight for histochemical, biochemical, and ultrastructural analyses. The majority of the soleus and extensor digitorum longus fibers exhibited simple cell shrinkage. However, approximately 1% of the fibers in flight soleus muscles appeared necrotic. Flight muscle fibers showed increased glycogen, lower subsarcolemmal staining for mitochondrial enzymes, and fewer subsarcolemmal mitochondria. During atrophy, myofibrils were eroded by multiple focal losses of myofilaments; lysosomal autophagy was not evident. Tripeptidylaminopeptidase and calcium-activated protease activities of flight soleus fibers were significantly increased, implying a role in myofibril breakdown. Simple fiber atrophy appears to account for muscle weakening during spaceflight, but fiber necrosis is also a contributing factor.