Automated rodent in situ muscle contraction assay and myofiber organization analysis in sarcopenia animal models.

Age-related sarcopenia results in frailty and decreased mobility, which are associated with increased falls and long-term disability in the elderly. Given the global increase in lifespan, sarcopenia is a growing, unmet medical need. This report aims to systematically characterize muscle aging in preclinical models, which may facilitate the development of sarcopenia therapies. Naïve rats and mice were subjected to noninvasive micro X-ray computed tomography (micro-CT) imaging, terminal in situ muscle function characterizations, and ATPase-based myofiber analysis. We developed a Definiens (Parsippany, NJ)-based algorithm to automate micro-CT image analysis, which facilitates longitudinal in vivo muscle mass analysis. We report development and characterization of translational in situ skeletal muscle performance assay systems in rat and mouse. The systems incorporate a custom-designed animal assay stage, resulting in enhanced force measurement precision, and LabVIEW (National Instruments, Austin, TX)-based algorithms to support automated data acquisition and data analysis. We used ATPase-staining techniques for myofibers to characterize fiber subtypes and distribution. Major parameters contributing to muscle performance were identified using data mining and integration, enabled by Labmatrix (BioFortis, Columbia, MD). These technologies enabled the systemic and accurate monitoring of muscle aging from a large number of animals. The data indicated that longitudinal muscle cross-sectional area measurement effectively monitors change of muscle mass and function during aging. Furthermore, the data showed that muscle performance during aging is also modulated by myofiber remodeling factors, such as changes in myofiber distribution patterns and changes in fiber shape, which affect myofiber interaction. This in vivo muscle assay platform has been applied to support identification and validation of novel targets for the treatment of sarcopenia.

The discovery of tetrahydrofluorenones as a new class of estrogen receptor beta-subtype selective ligands.

Synthesis and derivatization of a series of substituted tetrahydrofluorenone analogs giving potent, ERbeta subtype selective ligands are described. Several analogs possessing ERbeta binding affinities comparable to 17beta-estradiol but with greater than 75-fold selectivity over ERalpha are reported.

Distinct effects of the antiestrogen Faslodex on the stability of estrogen receptors-alpha and -beta in the breast cancer cell line MCF-7.

The effects of estrogen receptor (ER) ligands on the stability and transcriptional activity of ERbeta in the breast cancer cell lines MCF-7 and HeLa were examined. We found that ERbeta was degraded in the presence of 17beta-estradiol. Tamoxifen and Faslodex (ICI 182,780) prevented ERbeta receptor destabilization. In contrast to ERalpha, ERbeta degradation was not abolished by inhibitors of the proteasome-mediated protein degradation pathway. Furthermore, single point mutations in helix 12 of the receptor dramatically affected the stability and subsequent transcriptional activation of ERbeta.

Sensory ganglionectomy: theory, technical aspects, and clinical experience.

OBJECT: Sensory ganglionectomy offers theoretical advantages over rhizotomy but remains controversial because reported success rates vary widely. The authors sought to add to the available data on this subject and to review technical aspects of the surgery. METHODS: This retrospective chart review included 19 patients, in whom 22 operations were performed and 35 sensory ganglia were resected between May 1995 and May 1999. The eight women and 11 men ranged in age from 27 to 75 years (median age 40 years, average age 42.3 years). All patients had undergone extensive therapy and a mean of 2.4 previous operations (median three, range zero-eight operations) for their pain, all without long-term pain relief. Duration of symptoms varied, from 1 month (for the cancer patient) to 15 years (mean 5.9, median 4 years). Preoperatively, all patients underwent diagnostic selective nerve root blocks, which temporarily relieved their targeted pain. The duration of follow up averaged 22 months (median 13, range 1.5 [to death of the cancer patient]-58 months). Before undergoing the first ganglionectomy, nearly all patients rated their targeted pain as 8 to 10 (average 9.6, median 10) on an analog (0-10) pain scale. At 6 months all patients rated their ganglionectomy-specific pain as an average of 4.5 (median 4, range 0-8), and pain reduction of 50% or more was achieved in 74%. At 1 year or more the 13 patients available for study rated their pain as an average of 4.3 (median 4.5, range 0-9). There were no severe complications, residual pain was never worse than presurgical pain, and no patient experienced significant or lasting new motor deficits. CONCLUSIONS: Dorsal root ganglionectomy has a useful role in the treatment of a variety of refractory pain states, especially those involving radicular pain.

Bilateral anterior cingulotomy for chronic noncancer pain.

OBJECTIVE: To document the value of bilateral anterior cingulotomy for patients with intractable chronic noncancer pain. METHODS: Twenty-three patients who underwent 28 cingulotomies between 1979 and 1996 for chronic refractory pain were sent questionnaires regarding their subjective response to the surgery and its impact on their pain. Questions dealt with pre- and postoperative pain, ability to resume work or usual activity, medications, family and social interactions, and overall benefit of cingulotomy. Results were compared with long-term (average, 8 yr) clinical follow-up. In 13 patients, pain was predominantly caused by lumbar adhesive arachnoiditis or "failed back." The remainder had venous occlusive disease, ischemic bilateral leg pain, phantom leg pain, postoperative neck pain, or atypical facial pain. RESULTS: Eighteen patients returned questionnaires; two patients died of unrelated causes. Seventy-two percent of patients reported improvement in their pain, 55% were no longer taking narcotics, 67% noted improvement in their family life, and 72% noted improvement in their social interactions. Fifty-six percent of patients reported that the cingulotomy was beneficial, and 28% returned to their usual activities or work. Thirty-nine percent of patients developed transient or well-controlled seizures. Five patients required a second cingulotomy, and one patient did well despite developing brain abscesses. Patient assessments corresponded closely with clinical assessments. CONCLUSION: Bilateral anterior cingulotomy is safe for patients with refractory chronic pain. Seizures reported in this series were well controlled with medication. More than half of all respondents thought they had a positive outcome and that cingulotomy was beneficial to them. There were no deaths related to the procedure.

Trigeminal nerve peripheral branch phenol/glycerol injections for tic douloureux.

OBJECT: Trigeminal neuralgia or tic douloureux is a disease affecting older individuals, and thus, office-based "minimally invasive" therapy is inherently attractive. The author sought to determine whether injection of peripheral trigeminal branches with neurolytic solutions offers a simple, less invasive therapy, with low risk for patients with one- or two-division trigeminal neuralgia that is unresponsive to pharmacotherapy. METHODS: This retrospective study focused on a review of case charts from 18 patients treated for tic douloureux. Sixty injections of 10% phenol in glycerol were given to the 18 patients, six of whom had undergone other neurosurgical procedures. The median patient age was 74 years, ranging from 36 to 94 years. There were nine women and nine men. Forty-six injections were administered into the infraorbital nerve in its canal in the midface, 11 percutaneous injections were administered into the mandibular nerve just proximal to the mandibular canal in the ramus of the jaw, and three injections were administered into supraorbital nerves. Eighty-seven percent of injections brought marked or total relief initially. Of those injections that provided initial relief, 37% still provided relief after 1 year and 30% after 2 years, with relief lasting for a median of 9 months after each injection. Most patients whose pain recurred after months of relief requested a repeated procedure, rather than undergo a ganglion nerve block procedure or open surgery. There were no serious complications or dysesthetic pain. Facial sensory loss generally recovered within 6 months and was well tolerated. CONCLUSIONS: Office-based injection of trigeminal branches is a useful technique for neurosurgeons who treat trigeminal neuralgia. It is easily repeated and can provide immediate pain relief of intermediate duration.

Structural basis of sympathetic-sensory coupling in rat and human dorsal root ganglia following peripheral nerve injury.

Tyrosine hydroxylase immunocytochemistry was used to reveal the sympathetic postganglionic axons that sprout to form basket-like skeins around the somata of some primary sensory neurons in dorsal root ganglia (DRGs) following sciatic nerve injury. Ultrastructural observations in rats revealed that these sprouts grow on the surface of glial lamellae that form on the neurons. Sciatic nerve injury triggers glial cell proliferation in the DRG, and the formation of multilamellar pericellular onion bulb sheaths, primarily around large diameter DRG neurons. We infer that these glia participate in the sprouting process by releasing neurotrophins and expressing growth supportive cell surface molecules. Many DRG cell somata, and their axons in intact nerves and nerve end neuromas, express alpha2A adrenoreceptors intracytoplasmically and on their membrane surface. However, sympathetic axons never make direct contacts with the soma membrane. The functional coupling known to occur between sympathetic efferents and DRG neurons must therefore be mediated by the diffusion of neurotransmitter molecules in the extracellular space. Sympathetic basket-skeins were observed in DRGs removed from human neuropathic pain patients, but the possibility of a functional relation between these structures and sensory symptoms remains speculative.

Nuclear receptors have distinct affinities for coactivators: characterization by fluorescence resonance energy transfer.

UNLABELLED: Ligand-dependent interactions between nuclear receptors and members of a family of nuclear receptor coactivators are associated with transcriptional activation. Here we used fluorescence resonance energy transfer (FRET) as an approach for detecting and quantitating such interactions. Using the ligand binding domain (LBD) of peroxisome proliferator-activated receptor (PPARgamma) as a model, known agonists (thiazolidinediones and delta12, 14-PGJ2) induced a specific interaction resulting in FRET between the fluorescently labeled LBD and fluorescently labeled coactivators [CREB-binding protein (CBP) or steroid receptor coactivator-1 (SRC-1)]. Specific energy transfer was dose dependent; individual ligands displayed distinct potency and maximal FRET profiles that were identical when results obtained using CBP vs. SRC-1 were compared. In addition, half-maximally effective agonist concentrations (EC59s) correlated well with reported results using cell-based assays. A site-directed AF2 mutant of PPARgamma (E471A) that abrogated ligand-stimulated transcription in transfected cells also failed to induce ligand-mediated FRET between PPARgamma LBD and CBP or SRC-1. Using estrogen receptor (ERalpha) as an alternative system, known agonists induced an interaction between ERalpha LBD and SRC-1, whereas ER antagonists disrupted agonist-induced interaction of ERalpha with SRC-1. In the presence of saturating agonist concentrations, unlabeled CBP or SRC-1 was used to compete with fluorescently labeled coactivators with saturation kinetics. Relative affinities for the individual receptor-coactivator pairs were determined as follows: PPARgamma-CBP = ERalpha-SRC-1 > PPARgamma-SRC-1 >> ERalpha-CBP. CONCLUSIONS: 1) FRET-based coactivator association is a novel approach for characterizing nuclear receptor agonists or antagonists; individual ligands display potencies that are predictive of in vivo effects and distinct profiles of maximal activity that are suggestive of alternative receptor conformations. 2) PPARgamma interacts with both CBP and SRC-1; transcriptional activation and coactivator association are AF2 dependent. 3) Nuclear receptor LBDs have distinct affinities for individual coactivators; thus, PPARgamma has a greater apparent affinity for CBP than for SRC-1, whereas ERalpha interacts preferentially with SRC-1 but very weakly with CBP.

High basal gastric acid secretion in somatostatin receptor subtype 2 knockout mice.

BACKGROUND & AIMS: Somatostatin receptor subtype 2 (sst2) agonists inhibit gastric secretion. The role of sst2 in the regulation of acid secretion was assessed using sst2 knockout mice and urethane to induce somatostatin release. METHODS: Acid secretion was monitored every 10 minutes by gastric perfusion and backtitration of perfusates in fasted, urethane-anesthetized C57/129 sst2 (-/-) mice and wild-type (+/+) mice. The ileal vein was cannulated for drug injection. Intragastric pH and serum gastrin were monitored 1 hour after anesthesia without perfusion. RESULTS: Gastric pH values were lower in sst2 (-/-) mice (3.8 +/- 0.3) than in wild-type mice (7.1 +/- 0.1, P < 0.05), and there was no difference in gastrin levels. Basal acid output per 2 hours was 10-fold higher in sst2 knockout mice compared with wild-type mice. The gastrin antibody abolished the high basal acid secretion in sst2 (-/-) mice and had no effect in wild-type mice. The somatostatin antibody increased basal secretion by 4-fold in wild-type and had no effect in knockout mice. Somatostatin 14 or the sst2 agonist DC 32-87 inhibited pentagastrin-stimulated acid secretion in wild-type mice, but did not alter basal secretion in knockout mice. CONCLUSIONS: These results indicate that sst2 is the main subtype whereby endogenous somatostatin suppresses gastric acid secretion through inhibition of gastrin action.

Genetic and biochemical evidence for a novel avermectin-sensitive chloride channel in Caenorhabditis elegans. Isolation and characterization.

Avermectins are a class of macrocyclic lactones that is widely used in crop protection and to treat helminth infections in man and animals. Two complementary DNAs (GluClalpha and GluClbeta) encoding chloride channels that are gated by avermectin and glutamate, respectively, were isolated from Caenorhabditis elegans. To study the role of these subunits in conferring avermectin sensitivity we isolated a mutant C. elegans strain with a Tc1 transposable element insertion that functionally inactivated the GluClalpha gene (GluClalpha::Tc1). GluClalpha::Tc1 animals exhibit a normal phenotype including typical avermectin sensitivity. Xenopus oocytes expressing GluClalpha::Tc1 strain mRNA elicited reduced amplitude avermectin and glutamate-dependent chloride currents. Avermectin binding assays in GluClalpha::Tc1 strain membranes showed the presence of high affinity binding sites, with a reduced Bmax. These experiments suggest that GluClalpha is a target for avermectin and that additional glutamate-gated and avermectin-sensitive chloride channel subunits exist in C. elegans. We isolated a cDNA (GluClalpha2) encoding a chloride channel that shares 75% amino acid identity with GluClalpha. This subunit forms homomeric channels that are gated irreversibly by avermectin and reversibly by glutamate. GluClalpha2 coassembles with GluClbeta to form heteromeric channels that are gated by both ligands. The presence of subunits related to GluClalpha may explain the low level and rarity of target site involvement in resistance to the avermectin class of compounds.

Percutaneous radiofrequency upper thoracic sympathectomy.

Between June 1979 and May 1994, I performed 148 unilateral or bilateral sympathectomies on 247 limbs in 110 patients using a percutaneous radiofrequency technique, usually on an outpatient surgery basis. Patient ages ranged from 10 to 81 years, with 45 male and 65 female patients. Four patients had unsuccessfully undergone prior open surgical sympathectomy. Patients suffered from hyperhidrosis, vascular occlusion, Raynaud's disease or other chronic vasculopathies, painful causalgia or reflex sympathetic dystrophy, or Prinzmetal's angina. The sympathectomy technique has evolved over this 15-year period and is currently in its third phase. Changes in the procedure were based on anatomic and clinical/radiographic correlations and careful patient follow-up. Current modifications have reduced the frequency of both early and late failures. The present technique (Phase III) relies on neuroleptanalgesia with superficial local anesthesia only and does not require general anesthesia, intubation, or lung collapse. Two 18-gauge radiofrequency TIC needle electrodes (Radionics, Burlington, MA) are used. A series of three lesions is rostrocaudally made at each of the ganglion sites selected in an attempt to destroy the entire fusiform ganglion. Lesion sites are targeted by C-arm fluoroscopy and electrical stimulation, which produces a threshold of sensory awareness of > 1.0 V. Lesion effectiveness is monitored by bilateral finger plethysmography and hand skin temperature measurement. With the Phase III technique, the sympathetic activity in 96% of operated limbs after 2 years and in 91% of operated limbs after 3 years continues to be completely or largely interrupted. By comparison, I achieved similar success in 83 and 72% operated limbs with the Phase I technique and in 77 and 71% with the Phase II technique. Symptomatic pneumothorax, in six patients, has been the only serious complication. When necessary, a subsequent operation can easily be performed and is effective.

Spatial and temporal patterns of lin-12 expression during C. elegans hermaphrodite development.

The lin-12 gene encodes a receptor that mediates certain cell-cell interactions during Caenorhabditis elegans development. We have examined the expression of a lin-12::lacZ reporter gene in individual cells during the development of C. elegans hermaphrodites. lin-12::lacZ is expressed in a discrete spatial and temporal pattern during development and teh lin-12::lacZ reporter gene will provide a useful marker for other studies, particularly of somatic gonadal and vulval development. In general, the cells that express lin-12:: lacZ correspond to cells whose fates are known to be altered in lin-12 mutants implying that restriction of lin-12 expression may be an important regulatory mechanism; the exceptions to this statement may reveal the cellular defects that underlie aspects of the lin-12 phenotype that have not been previously explained. For decisions that are not naturally variable, lin-12::lacZ expression does not appear to change before or upon commitment to a cell fate implying that in these cases postranscriptional regulation of lin-12 activity may control cell fate specification.

Reciprocal changes in expression of the receptor lin-12 and its ligand lag-2 prior to commitment in a C. elegans cell fate decision.

During development of the C. elegans hermaphrodite gonad, two cells interact with each other, so that one chooses to become the anchor cell (AC) and the other becomes a ventral uterine precursor cell (VU). This interaction is mediated by the receptor LIN-12 and its apparent ligand LAG-2. We show that initially lin-12 and lag-2 are expressed in both cells, but prior to commitment, the expression patterns change in a reciprocal manner, so that lin-12 expression becomes restricted to the presumptive VU and lag-2 expression becomes restricted to the presumptive AC. In addition, lin-12 activity promotes expression of lin-12 and represses expression of lag-2. Furthermore, we show that positive autoregulation of lin-12 transcription in the presumptive VU is mediated by a cis-acting 5' regulatory sequence and is necessary to specify the VU fate. Our results suggest that transcriptional control is a component of the feedback mechanism involved in specifying the AC and VU fates.

Synergistic effect between intraneoplastic methotrexate and radiation on experimental intracerebral rat gliosarcoma.

Methotrexate (MTX) alone has a limited effect against malignant brain tumors, but we previously demonstrated a beneficial synergism between MTX and radiation therapy (XRT) against RT-9 gliosarcoma. Because the beneficial effects of that study were limited by systemic toxicity and poor brain penetration of MTX, we have continued our studies using direct intracerebral MTX therapy. Male CD-Fisher rats with intracerebrally implanted RT-9 gliosarcoma and indwelling brain tumor catheters were treated with intracerebral injections of MTX, whole-brain XRT, or a combination of both. MTX was given either as one of two "high-dose" treatments, on the basis of whole-body doses, or two "low-doses," on the basis of average brain weight. MTX alone at lower doses and XRT alone each prolonged survival moderately. High-dose MTX was highly toxic, but low-dose MTX was well tolerated. Combined MTX and XRT caused a significant prolongation of survival in all animals that survived treatment long enough to die from tumor growth.

glp-1 can substitute for lin-12 in specifying cell fate decisions in Caenorhabditis elegans.

Members of the lin-12/Notch gene family encode receptors for intercellular signals and are found throughout the animal kingdom. In many animals, the presence of at least two lin-12/Notch genes raises the issue of the significance of this duplication and divergence. In Caenorhabditis elegans, two lin-12/Notch genes, lin-12 and glp-1, encode proteins that are 50% identical, with different numbers of epidermal growth factor-like motifs in their extracellular domains. Many of the cell fate decisions mediated by lin-12 and glp-1 are distinct. Here, we express glp-1 protein under the control of lin-12 regulatory sequences in animals lacking endogenous lin-12 activity and find that glp-1 can substitute for lin-12 in mediating cell fate decisions. These results imply that the lin-12 and glp-1 proteins are biochemically interchangeable, sharing common ligand and effector proteins, and that the discrete lin-12 and glp-1 mutant phenotypes result from differential gene expression. In addition, these results suggest that the duplicate lin-12/Notch genes found in vertebrates may also be biochemically interchangeable.

Anterior communicating artery aneurysm rupture: an unusual cause of acute subdural hemorrhage.

A case of acute subdural hemorrhage caused by rupture of an anterior communicating artery aneurysm is presented. The patient is a young man who presented with an acute onset of neurologic symptoms; computed tomography revealed subdural hemorrhage in the absence of associated subarachnoid or intraparenchymal bleeding.

Intrathecal Depo-Medrol: a literature review.

Intrathecal methylprednisolone acetate (IT-MPA) treatments have been reported to be beneficial and safe for the treatment of low back problems and especially "failed back" problems, which include adhesive arachnoiditis. Other reports, however, have stressed the potential dangers of this treatment and have advised against its use. Many of these papers implicate the propylene glycol included in the methyl-prednisolone as being potentially harmful. Since the literature is rather extensive and clearly conflicting, it is difficult for those who treat patients with "failed back" problems to ascertain the risk/benefit ratio of this form of treatment, so a literature review and analysis has been undertaken. Published literature clearly attests to the usefulness and general safety of IT-MPA when used within certain limits. Although several studies implicate IT-MPA as a potential cause of arachnoiditis or other neurologic injury, most of the evidence is circumstantial and most complications followed multiple, large-dose, or frequent injections.