Long-term benefits of deflazacort treatment for boys with Duchenne muscular dystrophy in their second decade.

We compare the clinical course of 74 boys 10-18 years of age with Duchenne muscular dystrophy (DMD) treated (40) and not treated (34) with deflazacort. Treated boys were able to rise from supine to standing, climb stairs and walk 10 m without aids, 3-5 years longer than boys not treated. After 10 years of age, treated boys had significantly better pulmonary function than boys not treated and after 15 years of age, 8 of 17 boys not treated required nocturnal ventilation compared with none of the 40 treated boys. For boys over 15 years of age, 11 of 17 boys not treated required assistance with feeding compared to none of the treated boys. By 18 years, 30 of 34 boys not treated had a spinal curve greater than 20 degrees compared to 4 of 40 treated boys. By 18 years, 7 of 34 boys not treated had lost 25% or more of their body weight (treated 0 of 40) and 4 of those 7 boys required a gastric feeding tube. By 18 years, 20 of 34 boys not treated had cardiac left ventricular ejection fractions less than 45% compared to 4 of 40 treated boys and 12 of 34 died in their second decade (mean 17.6 +/- 1.7 years) primarily of cardiorespiratory complications. Two of 40 boys treated with deflazacort died at 13 and 18 years of age from cardiac failure. The treated boys were significantly shorter, did not have excessive weight gain and 22 of 40 had asymptomatic cataracts. Long bone fractures occurred in 25% of boys in both the treated and not treated groups. This longer-term study demonstrates that deflazacort has a very significant impact on health, quality of life and health care costs for boys with DMD and their families, and is associated with few side effects.

Proposed genitalia contouring guidelines in anal cancer intensity-modulated radiotherapy.

OBJECTIVE: Intensity-modulated radiotherapy (IMRT) for anal canal carcinoma (ACC) is associated with favourable toxicity outcomes. Side effects include sexual dysfunction, skin desquamation, pain and fibrosis to perineum and genitalia region. The genitalia are situated anterior to the primary ACC between two inguinal regions providing a challenging structure to avoid. Techniques improving outcomes require robust, consistent genitalia contouring to ensure standardization and production of fully optimized IMRT plans. Official recommendations for genitalia contouring are lacking. We describe a potential genitalia contouring atlas for ACC radiotherapy. METHODS: Following a review of genitalia CT anatomy, a contouring atlas was generated for male and female patients positioned prone and supine. Particular attention was paid to the reproducibility of the genitalia contour in all planes. RESULTS: Male and female genitalia positioned prone and supine are described and represented visually through a contouring atlas. Contoured areas in males include penis and scrotum, and in females include clitoris, labia majora and minora. The muscles, bone, prostate, vagina, cervix and uterus should be excluded. The genitalia contour extends laterally to inguinal creases and includes areas of fat and skin anterior to the symphysis pubis for both genders. CONCLUSION: This atlas provides descriptive and visual guidance enabling more consistent genitalia delineation for both genders when prone and supine. The atlas can be used for other sites requiring radiotherapy planning. ADVANCES IN KNOWLEDGE: This atlas presents visual contouring guidance for genitalia in ACC radiotherapy for the first time. Contouring methods provide reproducible genitalia contours that allow the provision of accurate dose toxicity data in future studies.

Purification and characterization of calcium/phospholipid-dependent kinase from adult human epidermis.

Tumor-promoting phorbol esters such as 12-0-tetradecanoylphorbol-13-acetate (TPA) cause epidermal inflammation and hyperplasia similar to that observed in psoriasis. Recent evidence suggests that these effects are mediated by a calcium/phospholipid-dependent protein kinase (protein kinase C), which is quantitatively the major cellular phorbol ester receptor. This report describes the partial purification and biochemical properties of this enzyme from adult human epidermis. Protein kinase C activity was purified 30-fold from high speed supernatants prepared from homogenates of keratome biopsies obtained from healthy volunteers. The partially purified preparation had a specific activity of 1.2 nmol/min/mg protein and an apparent molecular weight of 79,400. Activity was dependent on the presence of calcium and phosphatidylserine. At low calcium concentration (less than 0.1 mM) activity was greatly stimulated by 1,2-dioleoylglycerol. TPA mimicked the effect of diglyceride on enzyme activity, and the partially purified enzyme specifically bound phorbol dibutyrate (Kd = 2 nM). Protein kinase C activity was also present in the membrane fraction from adult human epidermis, and possessed properties similar to those of the cytosolic enzyme. We conclude that protein kinase C is present in human epidermis and is activated by TPA in a manner similar to that described for this enzyme from other tissues. These data lay the foundation for studying the role of protein kinase C in the regulation of epidermal growth and maturation.

Agonist-induced hydrolysis of phosphoinositides and formation of 1,2-diacylglycerol in adult human keratinocytes.

The ability of a variety of agonists to induce formation of inositol phosphates and 1,2-diacylglycerol in cultured adult human keratinocytes has been investigated. Histamine, bradykinin, and thrombin significantly stimulated formation of inositol mono-, bis-, and trisphosphate and 1,2-diacylglycerol within 5 min after addition. Aluminum fluoride also caused a dose-dependent accumulation of inositol phosphates suggesting the participation of a GTP binding protein in the regulation of phospholipase C-catalyzed phosphoinositide hydrolysis. These data demonstrate that human keratinocytes possess the capacity for phospholipase C-mediated signal transduction and suggest that this pathway may participate in the regulation of keratinocyte function.

Postischemic hypothermia fails to reduce ischemic injury in gerbil hippocampus.

The objective of this study was to determine whether postischemic hypothermia diminishes ischemic injury in gerbil hippocampus. Cerebral ischemia was produced by occluding both carotid arteries for 5 min, while maintaining the temperature of the cranium and rectum at 38 degrees C. Upon recirculation, the animals were divided into three groups: normothermic (38 degrees C), moderately hypothermic (33 degrees C), and deeply hypothermic (23 degrees C). In the normothermic group, cranial and rectal temperatures were maintained at 38 degrees C for 30 min and 2 h, respectively, prior to the removal of the temperature probes. In the moderately hypothermic group, cranial and rectal temperatures were reduced within 10 min to 33 degrees C for 1 h, and then rewarmed to 38 degrees C. In the deeply hypothermic group, rectal temperature was lowered within 10 min to 23 degrees C for 2 h prior to rewarming to 38 degrees C. After recovery for 1 week, the extent of histologic injury in the hippocampus was assessed in stained sections. Maximal injury was present in the CA1 subfield in all three groups. These results indicate that hippocampal injury was not diminished by postischemic hypothermia during the first 2 h of reperfusion. Thus, pharmacologic studies of postischemic protection in the gerbil model may not be strongly influenced by transient postischemic hypothermia.

Spreading depression induces tolerance of cortical neurons to ischemia in rat brain.

Cortical spreading depression (CSD) was induced in male Wistar rats by applying 2 M KCl to the frontal cortex of one hemisphere for 2 h. Saline was applied to the contralateral cortex in the same manner. Following recovery for 24 h, bilateral forebrain ischemia was induced for 6 min, and the animals were permitted to survive for 6 days for assessment of histopathology. The number of necrotic neurons was counted in the cerebral cortex, striatum, and hippocampus of both hemispheres. In separate sets of animals, the effects of KCl application on cortical direct current (DC) potential and regional expression of c-fos mRNA and 72-kDa heat shock protein (hsp72) mRNA were determined. Forebrain ischemia induced selective neuronal necrosis in both hemispheres, but the number of necrotic neurons in the cerebral cortex ipsilateral to the application of KCl was significantly smaller than that in the contralateral cortex (p < 0.02, Wilcoxon signed rank test, n = 7). In the striatum and hippocampus, there were no significant differences in neuronal necrosis between hemispheres. Application of KCl for 2 h induced 11 +/- 2 (mean +/- SD, n = 5) negative deflections of DC potential in the ipsilateral cortex; none were detected in the contralateral cortex. Widespread expression of c-fos mRNA was evident in the ipsilateral cortex, while hsp72 mRNA expression was restricted to the KCl application site. The present results demonstrate that CSD induces tolerance of cortical neurons to ischemia by mechanisms unrelated to hsp72.

Regional expression of heat shock protein-70 mRNA and c-fos mRNA following focal ischemia in rat brain.

In situ hybridization was used to estimate regional levels of heat shock protein-70 (HSP-70) mRNA and c-fos mRNA in two related models of focal cerebral ischemia. In the first model, permanent occlusion of the distal middle cerebral artery (MCA) alone caused a patchy increase in HSP-70 mRNA by 1 h in the central zone of the MCA territory of the ipsilateral neocortex. Tissue levels of HSP-70 mRNA continued to increase for several hours and remained elevated at 24 h. In contrast to the focal expression of HSP-70, c-fos mRNA was increased throughout the ipsilateral cerebral cortex by 15 min and remained elevated for least 3 h. The wide distribution of c-fos expression suggests it may have been caused by spreading depression. In the second model, severe focal ischemia was produced with a combination of transient (1-h) bilateral carotid artery occlusion and permanent MCA occlusion. Combined occlusion for 1 h without reperfusion caused expression of HSP-70 mRNA only in regions adjacent to the central zone of the MCA territory of the neocortex. However, reperfusion of the carotids for 2 h generated intense expression of HSP-70 mRNA throughout most of the ipsilateral cerebral cortex, white matter, striatum, and hippocampus. The wide-spread increase in HSP-70 mRNA suggests that reperfusion triggered expression in all previously ischemic regions. However, at 24 h of reperfusion, increased levels of HSP-70 mRNA were restricted primarily to the ischemic core of the neocortex. These results suggest that expression of HSP-70 mRNA is prolonged in regions undergoing injury, but is transient in surrounding regions that recover.

Mild hypothermia prevents ischemic injury in gerbil hippocampus.

The objective of this study was to define the degree of hypothermia required to diminish ischemic injury to CA1 hippocampal neurons following 5-min bilateral ischemia in the gerbil. The temperature of the body and head was regulated in three groups of animals at 37.5, 35.5, or 32.5 degrees C during 5-min bilateral carotid artery occlusion. Upon recirculation, normothermia was restored in all animals, and recovery was permitted for 1 week. Ischemic injury to CA1 hippocampus was determined using three endpoints: histologic injury, ATP content, and adenylate kinase activity. Reduction of head temperature to 35.5 and 32.5 degrees C during ischemia diminished histologic injury and improved CA1 levels of ATP and adenylate kinase activity in a dose-dependent manner. Indeed, 32.5 degrees C completely abolished ischemic injury to CA1 hippocampus, judging from each of the three endpoints. Reduction of head temperature to 32.5 degrees C delayed but did not prevent the depletion of ATP throughout the hippocampus during the 5-min ischemic insult. These results demonstrate that a decrease in head temperature of only 2 degrees C reduces the degree of CA1 injury in the gerbil model of 5-min bilateral ischemia. Thus, it is imperative to maintain strict normothermia in pharmacologic studies of ischemic protection. Finally, administration of nicardipine to normothermic gerbils failed to diminish ischemic injury in the CA1 hippocampus.

Regional expression of c-fos and heat shock protein-70 mRNA following hypoxia-ischemia in immature rat brain.

Cerebral ischemia induces the expression of a number of proteins that may have an important influence on cellular injury. The purpose of this study was to compare the regional effects of hypoxia-ischemia on the expression of the proto-oncogene, c-fos, and the heat shock protein-70 (HSP-70) gene in developing brain. Unilateral hypoxia-ischemia was produced in the brain of immature rats (7, 15, and 23 days after birth) using a combination of carotid artery ligation and systemic hypoxia (8% O2). After recovery for 2 and 24 h, the regional expression of c-fos and HSP-70 mRNA was determined using in situ hybridization. Littermates were permitted to recover for 1 week for assessment of histologic injury. Hypoxia-ischemia increased the expression of both c-fos and HSP-70 mRNA, but the topography of expression varied with the age of the animal as well as the mRNA species. In the 7-day-old group, expression of c-fos at 2 h increased in multiple regions of the ipsilateral hemisphere in nearly one-half of the animals, while HSP-70 mRNA was not expressed until 24 h and, then, predominantly in the hippocampus. In 15- and 23-day-old rats, expression of c-fos was increased at 2 h in the entorhinal cortex and in the dendritic field of the upper blade of the hippocampal dentate gyrus, while HSP-70 mRNA was prominently expressed in neocortex and the cell layers of the hippocampus. Interestingly, the strong expression of HSP-70 mRNA in dentate granule cells did not occur in the innermost layer of cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of cortical spreading depression on the levels of mRNA coding for putative neuroprotective proteins in rat brain.

Previous studies have demonstrated that cortical spreading depression (CSD) induces neuronal tolerance to a subsequent episode of ischemia. The objective of the present investigation was to determine whether CSD alters levels of mRNA coding for putative neuroprotective proteins. Unilateral CSD was evoked in male Wistar rats by applying 2 mol/L KCl over the frontal cortex for 2 hours. After recovery for 0, 2, or 24 hours, levels of several mRNA coding for neuroprotective proteins were measured bilaterally in parietal cortex using Northern blot analysis. Levels of c-fos mRNA and brain-derived neurotrophic factor (BDNF) mRNA were markedly elevated at 0 and 2 hours, but not 24 hours after CSD. Tissue plasminogen activator (tPA) mRNA levels were also significantly increased at 0 and 2 hours, but not 24 hours after CSD. Levels of the 72-kDa heat-shock protein (hsp72) mRNA were not significantly increased by CSD, except for a small elevation (20%) at 2 hours recovery. Levels of the 73-kDa heat-shock cognate (hsc73) mRNA were slightly, but significantly, increased at 2 and 24 hours of recovery. Finally, levels of mRNA for protease nexin-1 and glutamine synthetase were not significantly altered by CSD at any time studied. The current results support the hypothesis that neuronal tolerance to ischemia after CSD may be mediated by increased expression of FOS, BDNF, or tPA, but not by increased expression of hsp72, hsc73, nexin-1, or glutamine synthetase.

Regional alterations in glucose consumption and metabolite levels during postischemic recovery in cat brain.

Local CMRgl (LCMRgl) and metabolite levels were measured in the same tissue samples following 4 h of recirculation after 1 h of occlusion of the middle cerebral artery in the cat. The rate of glucose utilization was calculated using direct measurement of tissue deoxyglucose-6-phosphate and using a "lumped" constant corrected in each sample for alterations in tissue glucose. Increased LCMRgl (compared with that in sham-operated animals) occurred in regions with only minor alterations in levels of lactate and phosphocreatine. By contrast, LCMRgl was markedly depressed in regions with major changes in lactate and high-energy phosphates. Interestingly, tissue levels of glucose and unphosphorylated deoxyglucose were abnormally elevated in regions with profound energy failure. These results indicate an inhibition of glucose utilization in regions damaged by ischemia, despite the persistent elevation of tissue lactate. Increased glucose metabolism at 4 h post ischemia was detected only in areas with minor anaerobic alteration of metabolite levels.

Deflazacort in Duchenne muscular dystrophy: a comparison of two different protocols.

We compare the long-term benefits and side effects of deflazacort using two treatment protocols from Naples (N) and Toronto (T). Boys with Duchenne muscular dystrophy between the ages of 8 and 15 years and who had four or more years of deflazacort treatment were reviewed. Diagnostic criteria included males with proximal muscle weakness evident before 5 years, increased serum creatine kinase and genetic testing and/or a muscle biopsy consistent with Duchenne muscular dystrophy. Thirty-seven boys were treated with protocol-N using deflazacort at a dose of 0.6 mg/kg per day for the first 20 days of the month and no deflazacort for the remainder of the month. Boys with osteoporosis received daily vitamin D and calcium. Deflazacort treatment started between 4 and 8 years of age. Thirty-two were treated with protocol-T using deflazacort at a dose of 0.9 mg/kg per day, plus daily vitamin D and calcium. Treatment started between 6 and 8 years of age. All boys were monitored every 4-6 months. The results were compared with age-matched controls in the two groups (19 for protocol-N and 30 for protocol-T). For the boys treated with protocol-N, 97% were ambulatory at 9 years (control, 22%), 35% at 12 years (control, 0%), 25% at 15 years (control, 0%). For the 32 boys treated with protocol-T, 100% were ambulatory at 9 years (control, 48%), 83% at 12 years (control, 0%) and 77% at 15 years (control, 0%). No aids or leg braces were used for ambulation. In boys 13 years and older, a scoliosis of >20 degrees developed in 30% of the boys on protocol-N, 16% on protocol-T and 90% of controls. For protocol-N, no cataracts were observed while in protocol-T, 30% of boys had asymptomatic cataracts that required no treatment. Fractures occurred in 19% (control 16%) of boys on protocol-N and 16% (control, 20%) of boys on protocol-T. This report illustrates: (a) the importance of collaborative studies in developing treatment protocols in Duchenne muscular dystrophy and (b) the long-term beneficial effects of deflazacort treatment in both protocols. However, the protocol-T seems to be more effective and frequently is associated with asymptomatic cataracts.

Preconditioning with cortical spreading depression does not upregulate Cu/Zn-SOD or Mn-SOD in the cerebral cortex of rats.

Previous studies have demonstrated that preconditioning the brain with cortical spreading depression (CSD) induces tolerance to a subsequent episode of ischemia. In other models of preconditioning, induction of ischemic tolerance has been associated with increased expression of the antioxidant enzyme, superoxide dismutase (SOD). The objective of the present study was to determine whether CSD upregulates Cu/Zn-SOD or Mn-SOD. CSD was induced in one hemisphere by applying 2 M KCl to the frontal cortex in Wistar rats. After 2 or 24 h of recovery, Cu/Zn-SOD and Mn-SOD mRNA levels were determined in both hemispheres using Northern blot analysis. In separate rats, Cu/Zn-SOD and Mn-SOD protein levels were determined 24 and 72 h after CSD using Western blot analysis. In addition, total SOD, Cu/Zn-SOD and Mn-SOD enzymatic activities were measured 24 and 72 h after CSD using spectrophotometric and zymographic assays. At the times investigated, no significant differences in mRNA or protein levels for Cu/Zn-SOD or Mn-SOD were observed between the ipsilateral and contralateral cortex. Further, there were no significant differences in Cu/Zn-SOD or Mn-SOD enzymatic activities between the two hemispheres at 24 or 72 h after CSD. In addition, CSD did not alter the activities of Cu/Zn-SOD or Mn-SOD in either hemisphere, relative to those in unoperated animals. Taken together, these results fail to support the hypothesis that CSD-induced tolerance is mediated through the upregulation of Cu/Zn-SOD or Mn-SOD.

Dose-dependent induction of mRNAs encoding brain-derived neurotrophic factor and heat-shock protein-72 after cortical spreading depression in the rat.

Previous studies have demonstrated that cortical spreading depression (CSD) increases the expression of putative neuroprotective proteins. The objective of the present study was to elucidate the relationship between the number of episodes of CSD and steady-state levels of mRNAs encoding brain-derived neurotrophic factor (BDNF), heat-shock protein-72 (hsp72) and c-fos. Wistar rats were administered one, five, or twenty-five episodes of CSD evoked by application of 2 M KCl to the frontal cortex of one hemisphere. Animals were permitted to recover for 30 min, 2 h or 24 h prior to sacrifice. Total RNA was isolated from the parietal cortex of each hemisphere and analyzed using Northern blots. At 30 min recovery, levels of BDNF mRNA were not significantly elevated after 1 episode of CSD, but were increased 4-fold after five episodes of CSD and 11-fold after twenty-five episodes of CSD, relative to levels in the contralateral hemisphere. At 2 h recovery, BDNF mRNA levels increased 2-, 3- and 9-fold, respectively. At 24 h, BDNF mRNA had returned to control levels in all groups. Thus, CSD increased levels of BDNF mRNA in a dose-dependent fashion at the early recovery times. Hsp72 mRNA was below the level of detection after 1 and 5 episodes of CSD. However, after twenty-five episodes of CSD, hsp72 mRNA levels were increased in the ipsilateral hemisphere at 30 min and 2 h recovery. Unlike levels of BDNF and hsp72 mRNA, levels of c-fos mRNA were increased nearly to the same extent at 30 min and 2 h after one, five or twenty-five episodes of CSD before returning to control by 24 h recovery. These results demonstrate that CSD triggers a dose-dependent increase in the expression of genes encoding neuroprotective proteins, which may mediate tolerance to ischemia induced by CSD.

In vivo protein expression from mRNA delivered into adult rat brain.

The expression of proteins after local mRNA delivery has a great potential for analysis of protein function in vivo. To explore the feasibility of such a technique within the central nervous system (CNS), we delivered luciferase-encoding mRNA into the rat brain. The tissue distribution and stability of injected mRNA were analyzed using in situ detection and Northern hybridization, while luciferase expression was measured by enzymatic assay. Following intracerebral injection of lipofectin-complexed mRNA, expression of luciferase was detectable as early as 1 h, was maximal at 2-3 h, but was below the level of detection by 24 h. The extent of luciferase expression correlated with the amount of mRNA delivered. Luciferase expression was higher when lipofectin-complexed rather than naked mRNA was injected. In addition, the luciferase expression increased significantly by adding a 50 nt-long poly(A) tail to the 3'-end of the mRNA. Delivering mRNA to the cerebral cortex or hippocampus resulted in measurable luciferase activity at the injection sites but not in adjacent areas. Accordingly, the luciferase mRNA was also localized to the injection site, and the amount of intact transcript was significantly higher at 3 h compared to 24 h after injection. These results demonstrate that in vivo mRNA delivery is a feasible technique for immediate, transient overexpression of desired proteins in the CNS and, therefore, can serve as a model system to study the neurobiological effects of specific proteins.

Correlation of diffusion MRI and heat shock protein in a rat embolic stroke model.

The expression of 70 kDa heat shock protein (HSP-70) in focal ischemia occurs in regions that sustain sub-lethal ischemic injury, and may therefore be considered as a biological marker of the ischemic penumbra. In a rat embolic stroke model, using fibrin-rich emboli, we correlated the expression of HSP-70 mRNA with diffusion magnetic resonance imaging (MRI) to determine if HSP-70 mRNA expression was associated with alterations in the apparent diffusion coefficient (ADC) of brain tissue water, a putative early marker of cytotoxic injury that is readily measured in vivo. Serial ADC measurements were made for 120 min following embolic infarction in the right carotid artery territory. HSP-70 mRNA expression was observed at the boundaries of the densely ischemic zone, as judged by diffusion imaging. ADC values observed in HSP-70 mRNA-positive regions were intermediate between those observed in the ischemic core or in control regions. In addition, the volume of HSP-70 mRNA-positive tissue correlated positively with the volume of tissue showing intermediate ADC values at 120 min. These findings suggest that intermediate ADC values occur in penumbral regions. Heterogeneity of ischemic cellular injury is suggested as the basis for the intermediate ADC values observed in these regions.

Deflazacort treatment of Duchenne muscular dystrophy.

OBJECTIVE: We report the long-term effects on muscle strength and side effects with deflazacort in Duchenne muscular dystrophy (DMD). STUDY DESIGN: Boys with DMD between the ages of 7 and 15 years were reviewed retrospectively; 30 had been treated with deflazacort, and 24 had not. Muscle function, pulmonary function, and side effects were compared. RESULTS: The boys not treated with deflazacort stopped walking at 9.8 +/-1.8 years. Seven of 30 treated boys had stopped walking at 12.3+/-2.7 years (P<.05), and of the 23 boys who were still walking, 21 were older than 10 years. Pulmonary function (percent predicted functional vital capacity) was significantly greater in treated boys at 15 years (88% +/- 18%) than in boys not treated (39%+/-20%) (P<.001). Between 9 and 15 years, treated boys were shorter. Between 9 and 13 years, treated boys weighed less. After 13 years the treated boys maintained their weight, whereas boys not treated lost weight. Asymptomatic cataracts developed in 10 of 30 boys who received deflazacort. Other potential side effects of deflazacort such as hypertension, glucosuria, acne, infection, or bruising were not more common. CONCLUSIONS: We conclude that deflazacort can preserve gross motor and pulmonary function in boys with DMD with limited side effects.