Malignant astrocytoma with binucleated granular cells in a Sprague-Dawley rat.

A 2-year-old Sprague-Dawley rat with hindlimb paralysis was diagnosed with a cerebral malignant astrocytoma. The distinctive feature of this astrocytoma was the presence of scattered binucleated cells that contained hypereosinophilic, 1-2 micro m in diameter, cytoplasmic granules. The neoplastic astrocytes stained positively for vimentin (VIM), lysozyme, and phosphotungstic acid hematoxylin (PTAH). Within the binucleated cells, granules stained with PTAH and periodic acid-Schiff (PAS) before and after diastase digestion. Ultrastructurally, neoplastic astrocytes were characterized by cytoplasmic aggregates of electron-dense intermediate filaments consistent with VIM and desmin. The cytoplasm of binucleated cells contained numerous phagolysosomes enlarged by myelin figures and glycoprotein or glycolipid. Intermediate filaments were not present. This is the first description, in the rat, of a neoplasm with features resembling the human granular cell astrocytoma. Our findings suggest that an astrocytic origin should be considered for the binucleated cells in this neoplasm.

Myostatin expression in age and denervation-induced skeletal muscle atrophy.

Myostatin is hypothesized to regulate skeletal muscle mass and to be a potential target for therapeutic intervention in sarcopenia. To clarify whether myostatin is invariably associated with sarcopenia, this study examined the levels of expression of myostatin mRNA and protein in Sprague Dawley rats during aging- and denervation-induced sarcopenia. The level of myostatin mRNA in the gastrocnemius decreased progressively with age being 9, 34 and 56% lower at 6, 12 and 27 months, respectively, compared with mRNA levels at 1.5 months. In contrast, two low molecular mass isoforms of myostatin protein identified by Western blotting increased progressively with age. With denervation, myostatin mRNA was 31% higher on day 1 but by 14 days after sciatic neurectomy when the muscle had atrophied 50%, myostatin expression decreased 34% relative to the sham operated limb. Western analysis of the denervated gastrocnemius showed that myostatin protein levels varied in parallel with mRNA. These disparate patterns of expression of myostatin during age- and denervation-induced atrophy suggest that the regulation of myostatin is complex and variable depending on whether the atrophy is slowly or rapidly progressive.

Disparate dysfunction of skeletal muscles located near and distant from burn site in the rat.

This study tested the hypotheses that burn-induced change in muscle function varies at sites local and distant from burn and is related to changes in expression of acetylcholine receptors (AChRs) and muscle mass. In anesthetized rats, approximately 4% burn was inflicted over the tibialis anterior muscle of one limb. The contralateral leg served as control. In another study, a approximately 45% body surface area burn was produced on the trunk; controls were body sham-burned rats. The evoked twitch tensions of tibialis anterior muscles in both legs were measured together with AChR proteins and their transcripts. Compared with the contralateral leg, absolute tensions in the burned leg declined at days 1, 4, and 7 without loss of muscle mass so that tension per unit wet muscle mass (specific tension) decreased; at day 14, the tension decreased with muscle atrophy so that specific tension was unchanged. Membrane AChRs and/or the immature subunit transcript, AChRgamma messenger ribonucleic acid (mRNA) increased at days 4, 7, and 14, and both were inversely related to evoked tension (r =.43, P <.01 and r =.61, P <.0001, respectively). There was a direct correlation between AChR and AChRgamma mRNA (r =.82, P <.001), suggesting that the upregulated AChRs may contain the immature gamma-subunit isoform. After approximately 45% body burn, AChRs and mRNA did not change and the evoked tensions did not decline, but there was relative loss of muscle mass at days 7 and 14 so that specific tension increased. Burn trauma initially causes weakness of muscles directly under the burn, and this weakness may be partially related to increased expression of immature AChRs and later to muscle atrophy.

Long-term denervation impairs insulin receptor substrate-1-mediated insulin signaling in skeletal muscle.

Long-term denervation is associated with insulin resistance. To investigate the molecular bases of insulin resistance, the downstream signaling molecules of insulin receptor including insulin receptor substrate-1 (IRS-1) and phosphatidylinositol 3-kinase (PI 3-K) were examined in skeletal muscle of rats after 7 days of denervation. Long-term denervation attenuated insulin-stimulated activation of the initial steps of the intracellular signaling pathway. Insulin-stimulated tyrosine phosphorylation of insulin receptor was reduced to 36% (P < .005), as was the phosphorylation of IRS-1 to 34% (P < .0001) of control. While insulin receptor protein level was unchanged, the protein expression of IRS-1 was significantly decreased in denervated muscles. Insulin-stimulated percent tyrosine phosphorylation of IRS-1, normalized to the IRS-1 protein expression, was also reduced to 55% (P < .01) of control in denervated muscle. Denervation caused a decline in the insulin-induced binding of p85 regulatory subunit of PI 3-K to IRS-1 to 61% (P < .001) and IRS-1-associated PI 3-K activity to 57% (P < .01). These results provide evidence that long-term denervation results in insulin resistance because of derangements at multiple points, including tyrosine phosphorylation of insulin receptor and its downstream signaling molecule, IRS-1, protein expression of IRS-1, and activation of PI 3-K.

Thermal injury induces greater resistance to d-tubocurarine in local rather than in distant muscles in the rat.

UNLABELLED: We tested the hypothesis that resistance to d-tubocurarine (dTC) is more intense in muscles closer to, than distant from, burn, and is related to the expression of immature and total acetylcholine receptors (AChRs). Anesthetized rats received approximately 4% surface area burn over the tibialis muscle of one leg with the contralateral leg serving as control, or approximately 45% of the flank burn, with sham-burned pair fed controls. At 1, 4, 7, or 14 days later, the 50% effective dose of dTC, membrane AChRs, and messenger ribonucleic acid (mRNA) that encode the AChR gamma-subunit (AChRgamma-mRNA) were quantified in the tibialis. After the local leg burn, AChRs increased at Days 4, 7, and 14, and AChRgamma-mRNA at Days 4 and 7 after burn. The increased AChRgamma-mRNA correlated with total AChRs (r = 0.82), suggesting that the up-regulated AChRs may contain the immature isoform. The 50% effective dose of dTC after the local leg burn increased 1.2- to 1.5-fold at all periods and correlated significantly with AChRs (r = 0.54) and AChRgamma-mRNA (r = 0.57). After the flank burn, resistance was seen at Day 14 in association with muscle atrophy; AChRs and AChRgamma-mRNA were unaltered. The resistance to dTC after a local burn occurs sooner, is more marked, and is probably related to both increases and isoform changes in AChRs. The resistance at distant muscles appears unrelated to AChR changes. IMPLICATIONS: The resistance to d-tubocurarine after a burn differs between muscles near and distant from the burn and seems to depend on quantitative and qualitative changes in acetylcholine receptors and muscle atrophy associated with the insult.

Prednisolone-induced muscle dysfunction is caused more by atrophy than by altered acetylcholine receptor expression.

UNLABELLED: Large doses of glucocorticoids can alter muscle physiology and susceptibility to neuromuscular blocking drugs by mechanisms not clearly understood. We investigated the effects of moderate and large doses of prednisolone on muscle function and pharmacology, and their relationship to changes in muscle size and acetylcholine receptor (AChR) expression. With institutional approval, 35 Sprague-Dawley rats were randomly allocated to receive daily subcutaneous doses of 10 mg/kg prednisolone (P10 group), 100 mg/kg prednisolone (P100 group), or an equal volume of saline (S group) for 7 days. A fourth group of rats was pair fed (food restricted) with the P100 rats for 7 days (FR group). On Day 8, the nerve-evoked peak twitch tensions, tetanic tensions, and fatigability, and the dose-response curves of d-tubocurarine in the tibialis cranialis muscle were measured in vivo and related to muscle mass or expression of AChRs. Rate of body weight gain was depressed in the P100, FR, and P10 groups compared with the S group. Tibialis muscle mass was smaller in the P100 group than in the P10 or S groups. The evoked peak twitch and tetanic tensions were less in the P100 group than in the P10 or S groups, however, tension per milligram of muscle mass was greater in the P100 group than in the S group. The 50% effective dose of d-tubocurarine (microg/kg) in the tibialis muscle was smaller in the P10 (33.6 +/- 5.4) than in the S (61.9 +/- 5.0) or the P100 (71.3 +/- 9.6) groups. AChR expression was less in the P10 group than in the S group. The evoked tensions correlated with muscle mass (r(2) = 0.32, P < 0.001), however, not with expression of AChR. The 50% effective dose of d-tubocurarine did not correlate with muscle mass or AChR expression. Our results suggest that the neuromuscular dysfunction after prednisolone is dose-dependent, and derives primarily from muscle atrophy and derives less so from changes in AChR expression. IMPLICATIONS: The mechanisms by which chronic glucocorticoid therapy alters neuromuscular physiology and pharmacology are unclear. We suggest that the observed effects are dose-dependent and derive primarily from muscle atrophy and derive less from changes in acetylcholine receptor expression.

Fiber atrophy, but not changes in acetylcholine receptor expression, contributes to the muscle dysfunction after immobilization.

OBJECTIVES: Muscle weakness associated with critical illness can be due to the illness itself, immobilization associated with it, and/or to concomitant use of drugs that affect neuromuscular transmission. This study investigated the contribution of immobilization per se to the muscle dysfunction, as well as the associated morphologic and biochemical changes. DESIGN: Prospective, laboratory study. SETTING: Hospital research laboratory. SUBJECTS: Adult, male, Sprague-Dawley rats, weighing 200 to 250 g, were randomly allocated to three experimental groups, depending on the duration (7, 14, or 28 days) of limb immobilization (n = 9 to 11 per group) or sham immobilization (n = 5 to 6 per group). INTERVENTIONS: Chronic, unilateral immobilization (disuse) of the tibialis cranialis muscle was produced by fixing the knee and ankle joints at 90 degrees flexion. The contralateral unimmobilized leg and a separate group of sham-immobilized legs served as controls. MEASUREMENTS AND MAIN RESULTS: After 7, 14, or 28 days of disuse of the tibialis muscles, the peak isometric twitch (Pt) and tetanic (Po) tensions, as well as fatigability during 5 secs of nerve stimulation at 50, 100, and 150 Hz, were measured simultaneously in situ in the immobilized group and in its contralateral control, and in the sham-immobilized group and in its contralateral control. Muscle fiber and endplate morphologies were determined by histochemical methods; membrane acetylcholine receptors (AChRs) were determined by 125I alpha-bungarotoxin assay; and the level of expression of AChR subunit transcripts was determined by reverse transcriptase-polymerase chain reaction. Immobilization reduced Pt, Po, fatigability, muscle mass, and fiber cross-sectional area (p<.001 vs. controls), but did not decrease tension per unit muscle mass, fiber oxidative capacity, or motor endplate size. Muscle mass correlated with fiber cross-sectional area. Changes in fiber cross-sectional area accounted for 23% and 46% (p< or =.043) of the variability in Pt and Po, respectively. Pt and Po correlated poorly with total AChR protein and expression of epsilon- and gamma-subunit messenger RNA. CONCLUSION: To the extent that the immobilization model simulates the disuse-induced muscle dysfunction of critical illness, the results suggest that disuse per se may contribute to the muscle weakness, and that the muscle weakness is explained, almost exclusively, by the fiber atrophy and not by the qualitative or quantitative changes in AChR expression.

Morphological correlates of the differential responses of muscles to vecuronium.

We have noted previously that duration of vecuronium block correlated with fibre size in six muscle groups in the goat. Electrophysiological considerations suggest that the important factor should be the number of acetylcholine receptors (AChR) relative to fibre size. However, this hypothesis could not be verified in the goat because the number of AChR was relatively constant in the different muscles despite differences in fibre size. Therefore, in this study, we have investigated the relationship between sensitivity to vecuronium, as reflected by the ED50 and duration of block, of six muscles in the cat and the number of AChR per unit fibre cross-sectional area (CSA). The ED50 and duration of action (time to 50% recovery of the first twitch after a dose of 15 micrograms kg-1) of vecuronium in the tibialis cranialis, soleus, rectus abdominis, masseter, diaphragm and thyroarytenoideus muscles were determined during train-of-four stimulation and EMG recording in seven cats anaesthetized with pentobarbital. CSA of the muscle fibres and number of junctional AChR in these muscles were measured by histological methods and 125I-alpha-bungarotoxin binding assay, respectively, and the number of AChR per unit fibre CSA calculated. The association between muscle response (ED50 and duration of block) and fibre CSA or number of AChR per unit fibre CSA was then tested by regression analyses. Duration of block varied between the six muscles (mean 8.9 (SEM 2.6) to 20.3 (3.1) min; P = 0.0001) but ED50 did not (7.5 (1.5) to 15.6 (2.5) micrograms kg-1; P = 0.185). Fibre CSA and number of AChR per unit fibre CSA also varied between these muscles (P = 0.0001). Duration to 50% TI recovery was prolonged in muscles with a low number of AChR relative to fibre CSA (r2 = 0.30; P = 0.0002) and the ED50 increased as the number of AChR per fibre CSA increased (r2 = 0.240; P = 0.0016). These results in the cat suggest that the number of junctional AChR relative to fibre CSA is a morphological predictor of the differential sensitivities of muscles to neuromuscular blocking agents.

Mechanisms for the paradoxical resistance to d-tubocurarine during immobilization-induced muscle atrophy.

This study investigated whether immobilization-induced hyposensitivity to d-tubocurarine (dTC), up-regulation of acetylcholine receptors (AChRs) and changes in fiber size and motor endplate size persist indefinitely and whether they are causally related. Unilateral disuse of the tibialis muscle was produced in adult rats by pinning the knee and ankle joints at 90 degrees flexion. The contralateral unpinned and a separate group of sham-pinned legs served as controls. After 7, 14 or 28 days of disuse, the in vivo dose of dTC that produced 50% depression of nerve-evoked twitch (ED50) in the tibialis muscle increased 3.0-, 3. 2- and 2.1-fold (P < .05), and membrane AChRs increased 6.0- (P < . 05), 6.3- (P > .05) and 1.2-fold (P > .395) relative to control, respectively. Disuse caused muscle fiber atrophy (P < .01) but did not affect endplate size. Hence, the ratio of endplate size to fiber size increased. There was a transient increase in gene expression of all (including de novo expression of the gamma) subunits of the AChR, peaking at day 7 and returning to normal by day 28 of immobilization. The ED50 of dTC correlated directly with AChRs (R2 = 0.51; P < .0001) or the ratio of endplate size to fiber size (R2 = 0. 30; P < .001), and inversely with fiber size (R2 = 0.43, P < .0001). It is proposed that acting together, but not singly, the changes in AChRs, fiber size and relative endplate size contribute to the magnitude and time course of the resistance to dTC produced by chronic disuse.

Tacrine does not alter the potency of succinylcholine in the rat.

PURPOSE: Tacrine is a cholinesterase inhibitor used to manage Alzheimer's dementia. Given iv, it prolongs succinylcholine blockade in humans but the effects of chronic oral tacrine are not known. METHODS: Groups of adult rats were given 2.5 mg.kg-1 tacrine (chronic groups) or l ml saline (control) twice daily by gavage for one, two, four or eight weeks. An additional (acute) group received 2.5 mg.kg-1 tacrine iv. Twelve to 18 hr after the last gavage of tacrine or saline, and -20 min after iv tacrine, cumulative dose-response curves of succinylcholine were determined in the tibialis and soleus muscles in anaesthetized, ventilated rats during monitoring of evoked twitch response to indirect (nerve) train-of-four stimulation. RESULTS: The ED50 and ED95 of succinylcholine in control rats were (mean +/- SD) 204 +/- 41 and 382 +/- 96 micrograms.kg-1, respectively in the tibialis muscle, and 280 +/- 52 and 629 +/- 168 micrograms.kg-1 in the soleus muscle (P < 0.05 between muscles). In the acute and chronic tacrine groups, the mean ED50 and ED95 ranged from 166-197 and 277-396 micrograms.kg-1., respectively, in the tibialis muscle, and 248-333 and 546-667 micrograms.kg-1, in the soleus muscle. Dose responses did not differ among acute and chronic tacrine groups and the control group. CONCLUSION: Chronic oral tacrine does not alter muscle response to succinylcholine in the rat. This may not apply to Alzheimer patients receiving chronic tacrine since the interaction between acute tacrine and succinylcholine in the rat differs from that in humans.