Prognostic factors for cervical spondylotic amyotrophy: are signs of spinal cord involvement associated with the neurological prognosis?

OBJECTIVES: The purpose of this study was to clarify the prognostic factors for cervical spondylotic amyotrophy (CSA). METHODS: The authors retrospectively reviewed the medical records of 47 consecutive patients with CSA in whom the presence/absence of the pyramidal tract sign was noted. We analyzed whether the age, sex, presence of diabetes mellitus, medication (vitamin B12), type of the most atrophic and impaired muscle, the muscle strength at the presentation, the presence of the pyramidal tract sign, magnetic resonance imaging (MRI) findings, including the presence and number of T2 high signal intensity areas (T2 HIA) in the spinal cord and the conversion to surgery were associated with the recovery of muscle strength in the patients. In addition, we also investigated whether the duration of symptoms before surgery and the type of surgery were associated with the recovery of muscle strength in patients who required conversion to surgical treatment. RESULTS: The presence of T2 HIA on MRI (P=0.002), the number of T2 HIA on MRI (P=0.002) and conversion to surgery (P=0.015) were found to be significantly associated with a poorer recovery at the observational final follow-up. Further, the presence of the pyramidal tract sign (P=0.043) was significantly associated with a poor recovery at the final follow-up after surgery. CONCLUSION: The presence of a high signal intensity change on T2-weighted MRI and the pyramidal tract sign can be used as prognostic factors for patients with CSA.

A possible role for BDNF, NT-4 and TrkB in the spinal cord and muscle of rat subjected to mechanical overload, bupivacaine injection and axotomy.

Neurotrophins play a crucial role in the regulation of survival and the maintenance of specific functions for various populations of neurons. Neurotrophin-4 (NT-4) is most abundant in skeletal muscle, and is thought to promote sciatic nerve sprouting, inhibit agrin-induced acetylcholine receptor (AChR) clustering, evoke postsynaptic potentiation and induce mitochondrial proliferation. Using Western blot analysis, immunoprecipitation and immunohistochemistry, we investigated the distribution of NT-4 in slow- and fast-type muscles. We also tested the adaptive response of this protein in the mechanically overloaded muscle, in the regenerating muscle following bupivacaine injection and in the denervated muscle. Additionally, we investigated whether TrkB phosphorylation in the spinal cord and in the sciatic nerve occurs through the interaction with BDNF or NT-4 when the innervating muscle is damaged. Markedly more NT-4 was expressed in fast-type muscles compared with the slow types. TrkB protein was more frequently observed around the edge of myofibers (neuromuscular junction) of the soleus muscle compared with the gastrocnemius muscle. TrkB tyrosine phosphorylation occurred in the spinal cord but not in the sciatic nerve 24 h after bupivacaine injection of the innervating muscle. At the same time, the amount of TrkB co-precipitating with BDNF was markedly increased in the spinal cord. A rapid activation of TrkB (1-8 h) was also observed in the spinal cord after axotomy,while the amount of TrkB co-precipitating with NT-4 was markedly lower after axotomy. These results indicate that NT-4 is preferentially distributed in fast-type muscles. Furthermore, by interacting with BDNF and NT-4, the TrkB in the spinal cord may be important for the survival of motoneurons and outgrowth of injured peripheral axons following muscle damage.

Changes in dopamine transporter and c-Fos expression in the nucleus accumbens of alcohol-tolerant rats.

BACKGROUND: We have shown that neurochemical functions of 5-HT3 receptors in regulating dopamine (DA) release in the nucleus accumbens (ACC) after alcohol exposure compensate for the dysfunction of serotonergic activity to restore the original properties in processing alcohol tolerance, and that the development of alcohol dependence may be mediated by ACC 5-HT3 receptors. In the present study, the effects of chronic alcohol consumption on the functions of the dopamine transporter (DAT) and the expression of c-Fos proteins were investigated using in vivo brain microdialysis and immunocytochemistry. METHODS: Perfusion of cocaine and 1-(2-Bis-(4-fluorophenyl) methoxy) ethyl)-4-(3-phenylpropyl) piperizine (GBR 12909) through the microdialysis probe membrane increased the extracellular levels of DA in ACC of alcohol-treated rats that had developed alcohol tolerance by drinking 10% EtOH for 30 days. RESULTS: The magnitudes of DA reuptake or DAT inhibitors, cocaine, and GBR 12909 that induced DA availability in the ACC were significantly higher in alcohol-treated rats than in controls. When compared with control rats, the alcohol-treated rats exhibited higher levels of DA and its metabolite, DOPAC, in the ACC. Increased expression of the c-Fos-like protein was found in the ACC of alcohol-treated rats. These results show that (1) chronic alcohol consumption desensitizes or decreases the DAT of DA terminals in the ACC and that (2) EtOH causes cellular hyperexcitability of ACC dopaminergic neurons with increased Fos expression during alcohol tolerance. CONCLUSION: The findings suggested that an abnormality of the dopaminergic neurons in the ACC that are involved with DAT dysfunction is associated with the development of alcohol tolerance.

Rat and human membrane dipeptidase: tissue distribution and developmental changes.

Distribution and developmental changes in membrane dipeptidase activity were examined in rat and human tissues. The activity to hydrolyze glycyl-D-alanine in rat and human tissues was completely or almost completely inhibited by 5 mM cilastatin, suggesting that the activity was due to membrane dipeptidase and that the contribution of leucine aminopeptidase to the activity was minor. In 8-week-old rats, the activity was high in lung, kidney, pancreas and testis, and in each pooled sample of ileal mucosa, duodenal mucosa, jejunal mucosa and adrenal mucosa. A low activity was found in spleen, liver, serum and heart. The activity in lung, kidney, adrenal and intestinal mucosa increased up to the age of 5 or 8 weeks, while that in pancreas, testis and spleen reached a maximal level at around 3 weeks and declined thereafter. The distribution profile of the enzyme in postmortem tissues of adult humans was similar to that in rat, except for an extremely low activity in lung. The enzyme was also found in serum and urine from healthy volunteers. In urine, the activity was significantly correlated to the creatinine content. No clear dependence of the activity on gender or age was observed in urine and serum.

Effects of age and ethanol on dopamine and serotonin release in the rat nucleus accumbens.

Neural functions in the nucleus accumbens (ACC) play an important role in alcohol drinking behavior. In the present study, we observed the effects of age and ethanol (EtOH) on dopamine (DA) and serotonin (5-HT) release in the ACC of freely moving 4-, 10-, and 16-month-old rats using brain microdialysis techniques. After co-perfusion with 200 mM ethanol, ACC DA, and 5-HT release were decreased significantly in 16-month-old rats compared to those at 4 months old. ACC DA and 5-HT neurons of aged rats were less sensitive to ethanol. On the other hand, both basal extracellular DA and 5-HT release in the ACC were significantly higher in 16-month-old than in 4-month-old rats. Therefore, aging results in opposite changes in basal and alcohol-induced DA and 5-HT release in the ACC.

Downregulation of calpastatin in rat heart after brief ischemia and reperfusion.

The activities of calpain and its endogenous inhibitor, calpastatin, were measured in the soluble fraction of perfused rat heart after ischemia for 5-20 min and reperfusion for up to 30 min. The method for m-calpain measurement was modified: washing of the DEAE-cellulose column with 0.18 M NaCl instead of 0.15 M NaCl increased the m-calpain activity 12.5-fold. Ischemia for 20 min followed by reperfusion for 30 min did not affect the m-calpain activity but decreased the calpastatin activity. m-Calpain was enriched in the nucleus-myofibril fraction but was not further translocated on ischemia-reperfusion. Mu-calpain was below the limit of detection on immunoblotting or casein zymography, but its mRNA was substantially expressed, as detected on Northern blotting. Casein zymography also revealed a novel Ca2+-dependent protease without the typical characteristics of mu- or m-calpain. The immunoblotting of myocardial fractions showed that calpastatin was proteolyzed on ischemia-reperfusion. The calpastatin proteolysis was suppressed by a calpain inhibitor, Ac-Leu-Leu-norleucinal. Calpastatin may sequester calpain from its substrates in the normal myocardium, but may be proteolyzed by calpain in the presence of an unidentified activator in the early phase of calpain activation during ischemia-reperfusion, resulting in the proteolysis of calpastatin and then other calpain substrates.

Effects of ethanol on the levels of brain 6R-L-erythro-5, 6, 7, 8-tetrahydrobiopterin in the inbred strains of mice. DBA/2J, C3H/HeJ and C57BL/6J with different alcohol preferences.

6R-L-erythro-5, 6, 7, 8-tetrahydrobiopterin (6R-BH4) is a coenzyme for tyrosine, tryptophan and phenylalanine hydroxylases, the former two of which are the initial and the rate-limiting enzymes in the biosynthesis of the catecholamines and serotonin, respectively. The present study was designed to determine the changes in concentrations of 6R-BH4 in striatum and midbrain of the inbred strains of mice, DBA/2J, C3H/HeJ and C57BL/6J, with different genetically determined alcohol preferences, following the injection of ethanol (EtOH). The intraperitoneal administration of EtOH (0, 1, 2 and 4 g/kg) significantly and dose-dependently reduced the levels of striatal and midbrain 6R-BH4 in DBA/2J mice with the lowest alcohol preference, and EtOH (4 g/kg, i.p.) reduced the level of striatal 6R-BH4 in C3H/HeJ with medium alcohol preference. Following the administration of EtOH (4 g/kg, i.p.), brain 6R-BH4 levels in C57BL/6J mice with high alcohol preference were lowered compared with the control group, but the difference did not reach statistic significance. EtOH has a tendency to reduce the brain 6R-BH4 levels in mice with lower alcohol preference or higher sensitivity to EtOH. Based on these findings, it was proposed that differences in alcohol drinking behavior in the inbred strains of mice was influenced by brain 6R-BH4.

Proteolysis of ankyrin and Na+/K(+)-ATPase in postmortem rat brain: is calpain involved?

Ankyrin links the fodrin-based cytoskeleton to membrane proteins such as Na+/K(+)-ATPase, thereby maintaining cellular integrity. Immunoblotting by antibody raised against erythrocyte ankyrin demonstrated the proteolysis of ankyrin, which was highly correlated with postmortem interval (0-24 h). Proteolysis in the postmortem brain generated the 160-kDa fragment with an identical size as the fragment formed after in vitro proteolysis by calpain. Although microM Ca2+ induced the proteolysis in the homogenate, the presence of mu-calpain was not demonstrated by immunoblotting using the antibody that reacts with large subunits both of mu- and m-calpains. Na+/K(+)-ATPase was also proteolyzed in the postmortem brain.

Effects of ion channel blockers on rapid postmortem changes in extracellular dopamine and serotonin levels in the rat nucleus accumbens.

In the present study, we used in vivo brain microdialysis to examine the effects of ion channel blockers tetrodotoxin (TTX), EGTA-free Ca2+ and verapamil on rapid postmortem changes in extracellular levels of dopamine (DA), serotonin (5-HT) and their metabolites dihydroxyphenylacetic acid (DOPAC) and 5-hydroxyindoleacetic acid (5-HIAA) in the ACC of freely moving rats. Extracellular ACC DA levels decreased following the perfusion of the three ion channel blockers in freely moving rats, and then, at death by cervical dislocation, maximum respective 220-, 60- and 90-fold increases were observed in the extracellular output of DA in animals treated with EGTA, verapamil and TTX, respectively. Also, ACC 5-HT decreased following perfusion with the three blockers in the freely moving rats, and then maximum increases of 80-, 30- and 45-fold in the extracellular output of 5-HT were observed at death in animals treated with EGTA, verapamil and TTX, respectively, compared to the baseline. Cervical dislocation-induced rapid postmortem changes were inhibited markedly by perfusion with CSF containing the CA2+ entry blocker verapamil. These observations suggested that rapid postmortem changes in ACC DA and 5-HT release were associated with the action of calcium ion channels and/or voltage gated channels in the CNS.

Possibility of 5-HT3 receptor involvement in alcohol dependence: a microdialysis study of nucleus accumbens dopamine and serotonin release in rats with chronic alcohol consumption.

The present study was performed to examine the involvement of serotonin-3 (5-HT3) receptors in the rat nucleus accumbens (ACC) in alcohol dependence. In alcohol-treated rats, perfusion of 40 mM K+ and 100 mM ethanol (EtOH) through the microdialysis probe increased the extracellular levels of ACC dopamine (DA), compared with controls. Perfusion of the serotonin (5-HT) uptake inhibitor sertlarine enhanced the extracellular levels of ACC 5-HT in both groups. Increased 5-HT availability in the synaptic clefts on the ACC further activated ACC DA release in the alcohol-treated rats, in comparison with controls. In the final experiments, perfusion of the 5.0 microM 5-HT3 receptor agonist 2-methyl-5-HT (2-Me-5-HT) through the microdialysis probe enhanced the extracellular levels of ACC DA. Magnitude of 2-Me-5-HT-induced DA release was significantly higher in alcohol-treated rats than in controls. On the other hand, 40 mM K(+)- and 100 mM EtOH-induced extracellular 5-HT release in alcohol-treated rats were markedly inhibited. These results show that (1) chronic alcohol intake increases the sensitivity of 5-HT3 receptors, (2) 5-HT3 receptors regulate DA release in the ACC, (3) the dopaminergic neuronal systems associated with 5-HT3 ionophore in the ACC were upregulated after chronic alcohol exposure, and (4) chronic alcohol intake desensitizes the serotonergic neuronal systems in rat ACC. These findings suggest that neurochemical functions of 5-HT3 receptors in regulating DA release in the ACC after alcohol exposure compensate for the dysfunction of serotonergic activity to restore the original properties in processing alcohol tolerance and that the development of alcohol dependence may be mediated by ACC 5-HT3 receptors.

Translocation of protein kinase C-alpha, delta and epsilon isoforms in ischemic rat heart.

To explore the spatial and temporal localization of PKC isoforms during ischemia, we quantified PKC isoforms in the subcellular fractions in perfused rat heart by immunoblotting using specific antibodies against PKC isoforms. PKCs-alpha and epsilon translocated from the 100000 x g supernatant (S, cytosolic) fraction to the 1000 x g pellet (PI, nucleus-myofibril) and the 1000-100000 x g pellet (P2, membrane) fractions during 5-40 min of ischemia. PKC-delta redistributed from the P2 to the S fraction. A 50-kDa fragment of PKC-alpha appeared during ischemia possibly through calpain action. Immunohistochemical observations showed the different localizations of PKC-alpha, delta, and epsilon in the myocytes. The PKC assay displayed high basal levels of Ca(2+)-independent PKC, the activation of Ca(2+)-dependent PKC in the P1 and P2 fractions, and the activation of Ca(2+)-independent PKC in the P1 fraction after 20 min of ischemia. These observations show that ischemia induces different patterns of translocation of the three PKC isoforms, suggesting differences in their roles.

Involvement of calpain in postmortem proteolysis in the rat brain.

Calpain, a Ca(2+)-dependent neutral protease was examined to investigate its involvement in postmortem proteolysis in the rat brain. Western blotting analysis showed that the 240 kDa alpha-subunit of fodrin, a well-known substrate for calpain, was degraded to generate 150 kDa and 145 kDa fragments in the postmortem interval (0-24 h) at 25 +/- 3 degrees C. Postmortem proteolysis was dependent on ambient temperature. In in vitro experiments, the 150 kDa and 145 kDa fragments appeared in the homogenate with addition of Ca2+ (1 microM-1 mM) or in the microsomal fraction by incubation with purified calpain. Both calpain inhibitor-1 and leupeptin suppressed in vitro proteolysis. During the initial 0-24 h postmortem, the activity of m-calpain in the brain remained unaltered, while that of its endogenous inhibitor, calpastatin, decreased with the postmortem interval. These results indicate that calpain is involved in fodrin proteolysis in the postmortem rat brain. The ratio of the amount of the 150 kDa proteolytic product to that of the 240 kDa fodrin alpha-subunit was correlated significantly with the postmortem interval (0-16 h; r = 0.745).

Reperfusion of rat heart after brief ischemia induces proteolysis of calspectin (nonerythroid spectrin or fodrin) by calpain.

Rat myocardium expresses the 240- and 235-kD polypeptides antigenically related to alpha- and beta-subunits of brain calspectin (nonerythroid spectrin or fodrin), respectively. In the subcellular fractions of the myocardium, alpha-calspectin was found in the 600g, 10,000g, and 100,000g pellets, whereas beta-calspectin was localized to the 10,000g pellet. On the basis of the Na+,K(+)-ATPase activity and the contents of a gap junction protein, the sarcolemma was distributed to the 10,000g and 100,000g pellets, and the intercalated disks were enriched in the 10,000g pellet. Both alpha- and beta-calspectin were proteolyzed by calpain in vitro. The two subunits were also proteolyzed in vivo, when the rat hearts underwent 10 to 60 minutes of global ischemia followed by 30 minutes of reperfusion. The reperfusion following the ischemia induced the proteolysis of alpha-calspectin in the 10,000g and 100,000g pellets, producing the 150-kD fragment. A synthetic calpain inhibitor, calpain inhibitor-1, suppressed the degradation of calspectin in vivo, which indicates that calpain is responsible for the reperfusion-induced proteolysis of calspectin. The inhibitor also improved myocardial stunning. Immunohistochemical study revealed that the proteolysis of alpha-calspectin occurs at the intercalated disks and the sarcolemma after postischemic reperfusion, in accord with the biochemical data. These results suggest that degradation of calspectin partly accounts for the contractile failure of the myocardium after postischemic reperfusion by disrupting the membrane skeleton and the intercalated disks.

Cardiac arrest after traffic accident induced through vagal reflex in a case with bilateral stenosis of vertebral arteries.

An 84-year-old driver suffered cardiac arrest after a traffic accident. He was quickly resuscitated and transferred to a hospital where he was treated in a state of unconsciousness and respiratory failure for 20 days until his death. The brain stem was rendered anoxic during cardiac arrest, which caused the respiratory failure. Artificial ventilation and catecholamine infusion were carried out, resulting in myocardial degeneration. Bilateral stenosis of the vertebral arteries was disclosed, but no injuries or hemorrhage of the brain and spinal cord were detected. On days 3 and 4 after admission, immediately after the head of the victim was flexed forward for examinations, cardiac arrest was induced twice, but was controlled either by administering atropine or by restoring the original posture. Positional change is known to induce vagal reflex that results in bradycardia, hypotension or cardiac arrest in sensitive persons. The victim might have undergone the reflex-mediated cardiac arrest after the accident, to which the stenosis of the vertebral arteries may have contributed.

Death in head-down position: an autopsy report with reference to physiological mechanism.

A 74-year-old man was found dead in the head-down position in a hole in a pile of trash at his home. There was severe congestion and hemorrhage in the head and neck regions, and bronchial petechiae. Head-down tilt is a well-known model in studying the effects of weightlessness during space flight. By comparing our case with the physiological studies on head-down tilt, we determined that the head-down posture would increase cardiac output, and systemic and intracranial pressure, while it would suppress respiration by a central or peripheral mechanism.

Calpain is implicated in rat myocardial injury after ischemia or reperfusion.

Calpain activity was measured in the various subfractions of rat myocardia after global ischemia for 60 min or after ischemia followed by 30 min of reperfusion after the chromatographic separation of mu- and m-calpains. The activity of m-calpain after ischemia and that of mu-calpain after reperfusion were both higher than that in the control. The activity of the endogenous calpain inhibitor calpastatin in 10,000 x g supernatant was decreased after both ischemia and ischemia-reperfusion. The increase in m- and mu-calpain activities was suppressed by pre-ischemic perfusion with a synthetic calpain inhibitor, transepoxysuccinyl-L-leucylamido (4-guanidino) butane (E64d, 100 micrograms/ml). After reperfusion, the calpain activity in the 10,000 x g pellet was also increased, which was inhibited by pre-ischemic perfusion with E64d or dimethylsulfoxide (a solvent for E64d) or by reperfusion with 1 mmol/L ethyleneglycol bis (beta-aminoethylether)-N, N, N', N'-tetraacetic acid. SDS-polyacrylamide gel electrophoresis revealed the proteolysis of several proteins, including fodrin, in the 10,000 x g and 100,000 x g pellet fractions after ischemia and reperfusion, some of which were confirmed to be in vitro substrates of calpain. The creatine kinase release during the reperfusion was also partially inhibited by E64d or dimethylsulfoxide. Thus, calpain activity in the soluble or particulate fractions was altered during ischemia or reperfusion, and appeared to be implicated in the proteolysis of the membrane proteins, which may contribute to myocardial injury.

Successive cardiac death of brothers in association with stress.

A 38-year-old man (Case 1) suddenly collapsed while resting. His 54-year-old brother (Case 2) came soon thereafter began to perform cardiac massage and mouth-to-mouth resuscitation. However, the elder brother suddenly died after resuscitating his younger brother for about 30 minutes. Necropsy revealed an occlusive thrombus of the left anterior descending coronary artery and coagulation necrosis of the myocardium in Case 1. Case 2 was diagnosed as sudden cardiac death on the basis of the moderate coronary sclerosis and myocardial lesions including contraction band, fragmentation, waviness, bleeding, and mild subendocardial fibrosis. Pulmonary edema in the both cases revealed the presence of left ventricular heart failure. Case 2 is a good example of the contribution of psychological and physical stress to sudden cardiac death.