John T. Sladky

31P NMR studies in Duchenne muscular dystrophy: Age‐related metabolic changes

To evaluate possible progressive metabolic changes in Duchenne muscular dystrophy, we used 31P nuclear magnetic resonance spectroscopy to measure high-energy phosphate compounds and phosphorylated diesters (PDE) in resting gastrocnemius muscle of 14 Duchenne patients and 10 normal boys. The patients had higher inorganic phosphate (Pi), intracellular pH, and PDE; and lower phosphocreatine (PCr) and PCr/Pi ratio; ATP was not significantly different. The patients showed significant age-related decreases in PCr and PCr/Pi, and increases in Pi and PDE, but ATP did not change. In normal boys, ATP increased with age, but PCr and Pi did not. These studies imply progressive metabolic deterioration in Duchenne dystrophy.

Combined therapy with MK-801 and nimodipine for protection of ischemic brain damage.

Calcium ion can enter ischemic neurons through both receptor-operated and voltage-sensitive Ca2+channels. To attenuate this Ca2+ entry and Ca2+-induced neuronal injury, we tried a combined treatment with the noncompetitive N-methyl-D-aspartate (NMDA) antagonist, MK-801, and the dihydropyridine calcium antagonist, nimodipine, in a cat middle cerebral artery occlusion (1 hour) and reperfusion (3 hours) model. We measured changes in cytosolic free calcium, nicotinamide adenine dinucleotide/reduced nicotinamide adenine dinucleotide redox state, and blood flow in the cat cortex using a newly developed fluorometric technique with indo-1, a fluorescent intracellular Ca2+ indicator. The combined treatment, starting 5 minutes into ischemia, was effective in reducing both Ca2+ entry and histologic damage and in enhancing recovery of the electroencephalogram following reperfusion. MK-801 alone was also effective, but to a lesser extent. These data suggest that the dual blockade of Ca2+ entry using MK-801 and nimodipine may be a useful tool for protection against ischemic brain damage.

Dose‐dependent expression of neuronopathy after experimental pyridoxine intoxication

We examined the sequence of nervous system abnormalities that resulted when rats were given excess amounts of vitamin B6 (pyridoxine). High doses of pyridoxine (1,200 or 600 mg/kg/d) for 6 to 10 days caused a neuronopathy with necrosis of dorsal root ganglion (DRG) sensory neurons, accompanied by centrifugal axonal atrophy and breakdown of peripheral and central sensory axons. Large diameter neurons with long processes and large cytoplasmic volumes were especially affected. Smaller doses (300 to 150 mg/kg/d) for up to 12 weeks had minor effects on DRG neurons, but produced a neuropathy with axonal atrophy and degeneration. Guinea pigs given 1,800 mg/kg/d developed sensory neuronopathy, whereas mice given similar or higher doses did not have neuropathologic abnormalities. Multiple factors including rate of administration, differential neuronal vulnerability, and species susceptibility have bearing on the final expression of pyridoxine neurotoxicity.

TTX-sensitive and TTX-insensitive sodium channel mRNA transcripts are independently regulated in adult skeletal muscle after denervation

The expression of mRNA encoding the TTX-sensitive (SkM1) and TTX-insensitive (SkM2) voltage-dependent sodium channels in adult skeletal muscle is independently regulated. In normal skeletal muscle, only the SkM1 message is expressed and the level varies with muscle fiber type. After surgical denervation, the steady-state SkM1 mRNA level declines transiently, but returns to control levels within 5 days. Expression of SkM2 transcripts is markedly activated, reaching a peak 3 days after axotomy and then declining to a maintained level at approximately 30% of peak. Chemical denervation with botulinum toxin results in higher levels of SkM2 mRNA, which by 7 days posttreatment are 7-fold greater than levels in paired axotomized muscles. SkM2 expression subsequently declines as functional reinnervation appears. Quantal acetylcholine release appears to play a major role in suppression of SkM2 expression in adult innervated or reinnervated muscle, whereas nonquantal factors in toxin-treated, but not axotomized, muscle may sustain high level SkM2 mRNA expression.

brief communicationsL Corticosteroid‐responsive dominantly inherited neuropathy in childhood

We describe three children with corticosteroid-responsive inflammatory demyelinating polyneuropathy from families with dominantly inherited neuropathy. There were atypical clinical, electrophysiologic, and pathologic characteristics that suggested a coexistent inflammatory demyelinating neuropathy and that should alert the clinician to the possibility of an associated acquired, potentially treatable disorder.

Paternal transmission of congenital myotonic dystrophy.

We report a rare case of paternally transmitted congenital myotonic dystrophy (DM). The proband is a 23 year old, mentally retarded male who suffers severe muscular weakness. He presented with respiratory and feeding difficulties at birth. His two sibs suffer from childhood onset DM. Their late father had the adult type of DM, with onset around 30 years. Only six other cases of paternal transmission of congenital DM have been reported recently. We review the sex related effects on transmission of congenital DM. Decreased fertility of males with adult onset DM and contraction of the repeat upon male transmission contribute to the almost absent occurrence of paternal transmission of congenital DM. Also the fathers of the reported congenitally affected children showed, on average, shorter CTG repeat lengths and hence less severe clinical symptoms than the mothers of children with congenital DM. We conclude that paternal transmission of congenital DM is rare and preferentially occurs with onset of DM past 30 years in the father.

Acute improvement in histological outcome by MK-801 following focal cerebral ischemia and reperfusion in the cat independent of blood flow changes.

The present study reports on the acute effects of MK-801 on the histopathological outcome and blood flow changes during focal cerebral ischemia and reperfusion. In addition, acute changes in the EEG and blood pressure are also reported. In 16 halothane-anesthetized cats, the left middle cerebral artery (MCA) was occluded for 2 h followed by 4 h of reperfusion. Thirty minutes after the onset of ischemia, eight animals were treated with 1 mg/kg of MK-801, while eight animals received saline. Blood flow from the peripheral MCA territory was measured with H2 clearance. There was a comparable reduction in blood flow (down to 20% of control) in the ischemic gyri of the two groups followed by a partial recovery after recirculation. There was a similar decrease in the EEG amplitude over the ischemic central MCA territory in the treated and the untreated group. Treatment with MK-801 induced a burst suppression in the EEG and a transient drop (11.4 ± 6.5 mm Hg) in the mean arterial pressure. The volume of early ischemic damage decreased by one-third in the MK-801-treated group compared to the untreated one, both in the total hemisphere (from 29 ± 10 to 20 ± 5%) and in the hemispheric cortex (range 36 ± 8 to 24 ± 13%). A major fraction of this improvement was localized to the middle and posterior parietal (mainly perifocal) regions of the MCA territory. These results show that in our model, MK-801 improves histopathological outcome despite the lack of apparent effect on the cortical blood flow, and an adverse effect on the systemic blood pressure. This is the first report that describes data on a reproducible model of reperfusion after temporary occlusion of the MCA in a cat, extending the findings of the Glasgow group, who observed similar neuroprotection in models of permanent MCA occlusion.

The Effect of Hyperglycemia on Intracellular Calcium in Stroke

The effect of hyperglycemia on cytosolic free calcium ([Ca2+]i) during temporary focal cerebral ischemia was investigated in cats using a fluorometric technique. The middle cerebral artery (MCA) was occluded for a period of 1 h, after which the clip was removed. In seven animals, plasma glucose was raised to 500–700 mg/dl by infusion of a 50% glucose solution starting 30 min after MCA occlusion, while eight animals were kept normoglycemic during and following occlusion. MCA occlusion induced a significant, but identical, elevation of the [Ca2+]i signal ratio (400/506 nm) in both the normoglycemic group (from 1.40 to 1.97 ± 0.34, p < 0.01) and in the hyperglycemic group (from 1.40 to 2.00 ± 0.53, p < 0.01) at the end of the occlusion. Between 10 and 30 min after reopening, the [Ca2+]i signal ratio decreased to control levels in the normoglycemic group (1.40 ± 0.11 and 1.36 ± 0.08 at 10 and 30 min after reopening, respectively), but remained elevated in the hyperglycemic group (1.69 ± 0.18 and 1.65 ± 0.21 at 10 and 30 min after reopening, respectively). There was a statistically significant difference between the two groups (p < 0.01). These data suggest that hyperglycemia may be harmful to calcium recovery during the early recirculation period following focal cerebral ischemia.

Effect of superoxide dismutase on intracellular calcium in stroke.

To clarify the relationship between calcium metabolism and free radical damage during the reperfusion period following ischemia, we investigated the effect of superoxide dismutase (SOD) on changes in cytosolic free calcium, cortical blood flow, and histologic changes following focal cerebral ischemia and reperfusion in 12 cats. Using indo-1, a fluorescent intracellular Ca2+ indicator, we simultaneously measured changes in the Ca2 + signal ratio (400:500 nm), NADH signal (464 nm), and reflectance (340 nm) during ultraviolet excitation (340 nm) directly from the cortex in vivo. The middle cerebral artery (MCA) was occluded for 1 h; only cats in which the EEG amplitude was depressed to <10% of control during the occlusion were entered into the study. Starting 2 min prior to release of the occlusion and continuing for 4 min, SOD (10,000 U/kg) was slowly infused in six cats, while in six cats, the vehicle only was infused. During MCA occlusion, the Ca2+ signal ratio increased significantly in both groups with no significant difference between the groups. During reperfusion, the Ca2+ signal ratio remained at a high level in the vehicle-treated group, while in the SOD-treated group, the Ca2+ signal ratio decreased. There was a statistically significant difference between the two groups at 10, 20, and 30 min after reperfusion (p < 0.01). The histologically damaged area in the SOD-treated group was significantly smaller than that in the vehicle-treated group (p < 0.01). These data suggest that the histoprotective action of SOD may be due to its ability to attenuate increases in intracellular calcium during the recirculation period following focal cerebral ischemia.

Prolonged effects of MK-801 in the cat during focal cerebral ischemia and recovery: Survival, EEG activity and histopathology

Previously we reported an improvement in histological outcome in cats treated with MK-801 shortly after the induction of temporary middle cerebral artery occlusion, and examined after 2 h of ischemia followed by 4 h of reperfusion. This study investigates the prolonged effects of the same drug treatment. Focal cerebral ischemia was produced in 34 cats by temporary occlusion of the left middle cerebral artery for 2 h. Stroke severity was determined using the ratio of the EEG amplitude from the ipsilateral to that of the contralateral hemisphere. Thirty minutes after the onset of ischemia, cats were treated i.v. with either 1 mg/kg MK-801 or saline. Electrocortical activity of the animals who survived were followed for 6 days postocclusion at which point they were sacrificed for histopathological analysis. Twelve of the animals died during recovery, of which 4 were MK-801 treated, and 8 were saline controls. The EEG ratios in the non-surviving animals were more depressed than in the animals that survived, whereas the depression in the EEG amplitude in both the treated and the control surviving animals was equal. Among the survivors no reduction in infarct size with MK-801 treatment was observed. Thus treatment with MK-801 in the middle cerebral artery occlusion model in the cat leads to a significant increase in the rate of survival (P < 0.05), but no prolonged improvement in late histopathology, in contrast with acute histological findings using this model. MK-801 treatment may be shifting the stroke model towards the survival of animals with larger infarcts. Histological recovery during prolonged reperfusion may eliminate the early neuroprotective effects seen with MK-801 treatment.