Sacroplasty for symptomatic sacral hemangioma: a novel treatment approach. A case report.

Painful vertebral body hemangiomas have been successfully treated with vertebroplasty and kyphoplasty. Sacral hemangiomas are uncommon and as such painful sacral hemangiomas are rare entities. We report what we believe is only the second successful treatment of a painful sacral hemangioma with CT-guided sacroplasty. A 56-year-old woman with a history of right-sided total hip arthroplasty and lipoma excision presented to her orthopedic surgeon with persistent right-sided low back pain which radiated into her buttock and right groin and hindered her ability to walk and perform her activities of daily living. MRIs of the thoracic spine, lumbar spine and pelvis showed numerous lesions with imaging characteristics consistent with multiple hemangiomas including a 2.2×2.1 cm lesion involving the right sacrum adjacent to the right S1 neural foramen. Conservative measures including rest, physical therapy, oral analgesics and right-sided sacroiliac joint steroid injection did not provide significant relief. Given her lack of improvement and the fact that her pain localized to the right sacrum, the patient underwent CT-guided sacroplasty for treatment of a painful right sacral hemangioma. Under CT fluoroscopic guidance, a 10 gauge introducer needle was advanced through the soft tissues of the back to the margin of the lesion. Biopsy was then performed and after appropriate preparation, cement was then introduced through the needle using a separate cement filler cannula. Appropriate filling of the right sacral hemangioma was visualized using intermittent CT fluoroscopy. After injection of approximately 2.5 cc of cement, it was felt that there was near complete filling of the right sacral hemangioma. With satisfactory achievement of cement filling, the procedure was terminated. Pathology from biopsy taken at the time of the procedure was consistent with hemangioma. Image-guided sacroplasty with well-defined endpoints is an effective, minimally invasive and safe procedure. Patients with painful sacral hemangiomas can be treated with this technique with no significant complications.

Fixation of pathological humeral fractures by the cemented plate technique.

Pathological fractures of the humerus are associated with pain, morbidity, loss of function and a diminished quality of life. We report our experience of stabilising these fractures using polymethylmethacrylate and non-locking plates. We undertook a retrospective review over 20 years of patients treated at a tertiary musculoskeletal oncology centre. Those who had undergone surgery for an impending or completed pathological humeral fracture with a diagnosis of metastatic disease or myeloma were identified from our database. There were 63 patients (43 men, 20 women) in the series with a mean age of 63 years (39 to 87). All had undergone intralesional curettage of the tumour followed by fixation with intramedullary polymethylmethacrylate and plating. Complications occurred in 14 patients (22.2%) and seven (11.1%) required re-operation. At the latest follow-up, 47 patients (74.6%) were deceased and 16 (25.4%) were living with a mean follow-up of 75 months (1 to 184). A total of 54 (86%) patients had no or mild pain and 50 (80%) required no or minimal assistance with activities of daily living. Of the 16 living patients none had pain and all could perform activities of daily living without assistance. Intralesional resection of the tumour, filling of the cavity with cement, and plate stabilisation of the pathological fracture gives immediate rigidity and allows an early return of function without the need for bony union. The patient's local disease burden is reduced, which may alleviate tumour-related pain and slow the progression of the disease. The cemented-plate technique provides a reliable option for the treatment of pathological fractures of the humerus.

Atm is a negative regulator of intestinal neoplasia.

The ataxia telangiectasia-mutated (ATM) gene has been implicated as an early barrier to the growth and progression of incipient solid tumors. Here, we show that germ-line nullizygosity for the mouse Atm gene significantly increases the proliferative index, net growth rate and multiplicity of intestinal adenomas in two distinct models of familial colon cancer: Apc(Min/+) and Apc(1638N/+). These effects of Atm deficiency are quantitatively different from deficiency for either of the genomic stability genes Bloom's syndrome helicase or DNA ligase 4, and the effect of Atm loss on tumor multiplicity is largely independent of the effect of ionizing radiation. Furthermore, the loss of heterozygosity rates at the adenomatous polyposis coli (Apc) locus are unaffected by Atm loss. Taken together, these data implicate the Atm gene product as a barrier to dysplastic growth in the early stages of intestinal tumor progression, independent of its effects on genomic stability.

Two neuropeptides colocalized in a command-like neuron use distinct mechanisms to enhance its fast synaptic connection.

In many neurons more than one peptide is colocalized with a classical neurotransmitter. The functional consequence of such an arrangement has been rarely investigated. Here, within the feeding circuit of Aplysia, we investigate at a single synapse the actions of two modulatory neuropeptides that are present in a cholinergic interneuron. In combination with previous work, our study shows that the command-like neuron for feeding, CBI-2, contains two neuropeptides, feeding circuit activating peptide (FCAP) and cerebral peptide 2 (CP2). Previous studies showed that high-frequency prestimulation or repeated stimulation of CBI-2 increases the size of CBI-2 to B61/62 excitatory postsynaptic potentials (EPSPs) and shortens the latency of firing of neuron B61/62 in response to CBI-2 stimulation. We find that both FCAP and CP2 mimic these two effects. The variance method of quantal analysis indicates that FCAP increases the calculated quantal size (q) and CP2 increases the calculated quantal content (m) of EPSPs. Since the PSP amplitude represents the product of q and m, the joint action of the two peptides is expected to be cooperative. This observation suggests a possible functional implication for multiple neuropeptides colocalized with a classical neurotransmitter in one neuron.

A newly identified buccal interneuron initiates and modulates feeding motor programs in aplysia.

Despite considerable progress in characterizing the feeding central pattern generator (CPG) in Aplysia, the full complement of neurons that generate feeding motor programs has not yet been identified. The distribution of neuropeptide-containing neurons in the buccal and cerebral ganglia can be used as a tool to identify additional elements of the feeding circuitry by providing distinctions between otherwise morphologically indistinct neurons. For example, our recent study revealed a unique and potentially interesting unpaired PRQFVamide (PRQFVa)-containing neuron in the buccal ganglion. In this study, we describe the morphological and electrophysiological characterization of this novel neuron, which we designate as B50. We found that activation of B50 is capable of producing organized rhythmic output of the feeding CPG. The motor programs elicited by B50 exhibit some similarities as well as differences to motor programs elicited by the command-like cerebral-to-buccal interneuron CBI-2. In addition to activating the feeding CPG, B50 may act as a program modulator.

PRQFVamide, a novel pentapeptide identified from the CNS and gut of Aplysia.

We have purified a novel pentapeptide from the Aplysia nervous system using bioassay on gut contractions. The structure of the peptide is Pro-Arg-Gln-Phe-Val-amide (PRQFVa). The precursor for PRQFVa was found to code for 33 copies of PRQFVamide and four related pentapeptides. Peaks corresponding to the predicted masses of all five pentapeptides were detected in Aplysia neurons by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Northern analysis revealed that expression of the precursor is abundant in the abdominal ganglion, much less in the pedal and cerebral ganglia, and rarely seen in the buccal and pleural ganglia. PRQFVa-positive neurons, mapped by immunohistochemistry and in situ hybridization, were present in all the central ganglia. PRQFVa immunopositive processes were observed in the gut, particularly in association with the vasculature. Some arteries and other highly vascularized tissues, such as the gill and the kidney, also contain numerous PRQFVa immunopositive processes. Application of synthetic PRQFVa suppresses not only contractions of the gut but also contractions of vasculature. PRQFVa is expressed in some of the neurons within the feeding circuitry and application of synthetic PRQFVa was found to decrease the excitability of some (B4/5 and B31/32) but not all (B8) neurons of the buccal feeding circuit. Our findings suggest that PRQFVa may act as a modulator within the feeding system as well as in other systems of Aplysia.

Identification and characterization of the feeding circuit-activating peptides, a novel neuropeptide family of aplysia.

We use a multidisciplinary approach to identify, map, and characterize the bioactivity of modulatory neuropeptides in the circuitry that generates feeding behavior in Aplysia. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry of the cerebral-buccal connective (CBC), a nerve containing axons of many interneurons that control feeding behavior of Aplysia, was used to identify neuropeptides that may participate in generation and shaping of feeding motor programs. Using this functionally oriented search, we identified a novel family of peptides that we call the feeding circuit-activating peptides (FCAPs). Two peptides with masses identical to those observed in the CBCs (molecular weight 1387 and 1433) were purified from buccal ganglia and partially sequenced using mass spectrometry. The amino acid sequence was then used to clone the FCAP precursor, which encodes multiple copies of eight different FCAPs. The two FCAPs present in highest copy number correspond to those observed in the CBC. The distribution of FCAP expression was mapped using Northern analysis, whole-mount in situ hybridization, and immunocytochemistry. Consistent with our initial findings, FCAP-immunopositive axons were observed in the CBC. Furthermore, we found that FCAP was present in some cerebral-buccal and buccal-cerebral interneurons. As their name suggests, FCAPs are capable of initiating rhythmic feeding motor programs and are the first neuropeptides with such activity in this circuit. The actions of FCAPs suggest that these peptides may contribute to the induction and maintenance of food-induced arousal. FCAPs were also localized to several other neuronal systems, suggesting that FCAPs may play a role in the regulation of multiple behaviors.

Motor program selection in simple model systems.

In recent years, studies of molluscan and crustacean feeding circuits have greatly expanded our knowledge of how the nervous system selects specific behaviors. Increasing use of neurobehavioral studies, and examination of the roles of identified command-like or influential neurons have narrowed the gap between knowledge of circuit connectivity and understanding of the normal behavioral functions of these circuits.

The enterins: a novel family of neuropeptides isolated from the enteric nervous system and CNS of Aplysia.

To identify neuropeptides that have a broad spectrum of actions on the feeding system of Aplysia, we searched for bioactive peptides that are present in both the gut and the CNS. We identified a family of structurally related nonapeptides and decapeptides (enterins) that are present in the gut and CNS of Aplysia, and most of which share the HSFVamide sequence at the C terminus. The structure of the enterin precursor deduced from cDNA cloning predicts 35 copies of 20 different enterins. Northern analysis, in situ hybridization, and immunocytochemistry show that the enterins are abundantly present in the CNS and the gut of Aplysia. Using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry we characterized the enterin-precursor processing, demonstrated that all of the precursor-predicted enterins are present, and determined post-translational modifications of various enterins. Enterin-positive neuronal somata and processes were found in the gut, and enterins inhibited contractions of the gut. In the CNS, the cerebral and buccal ganglia, which control feeding, contained the enterins. Enterin was also present in the nerve that connects these two ganglia. Enterins reduced the firing of interneurons B4/5 during feeding motor programs. Such enterin-induced reduction of firing also occurred when excitability of B4/5 was tested directly. Because reduction of B4/5 activity corresponds to a switch from egestive to ingestive behaviors, enterin may contribute to such program switching. Furthermore, because enterins are present throughout the nervous system, they may also play a regulatory role in nonfeeding behaviors of Aplysia.

Neural mechanisms of motor program switching in Aplysia.

The Aplysia multifunctional feeding central pattern generator (CPG) produces at least two types of motor programs, ingestion and egestion, that involve two sets of radula movements, protraction-retraction and opening-closing movements. In ingestion, the radula closes during retraction to pull food in, whereas in egestion, the radula closes during protraction to push inedible objects out. Thus, radula closure shifts the phase in which it occurs with respect to protraction-retraction in the two programs. To identify the central switching mechanisms, we compared activity of CPG neurons during the two types of motor programs elicited by a higher-order interneuron, cerebral-buccal interneuron-2 (CBI-2). Although CPG elements (B63, B34, and B64) that mediate the protraction-retraction sequence are active in both programs, two other CPG elements, B20 and B4/5, are preferentially active in egestive programs and play a major role in mediating CBI-2-elicited egestive programs. Both B20 and B4/5 control the phasing of radula closure motoneurons (B8 and B16) to ensure that, in egestive programs, these motoneurons fire and produce radula-closing movements only during protraction. Elsewhere, another higher-order interneuron, CBI-3, was shown to convert CBI-2-elicited egestion to ingestion. We show that CBI-3 switches the programs by suppressing the activity of B20 and B4/5. CBI-3, active only during protraction, accomplishes this through fast inhibition of B20 during protraction and slow inhibition of B4/5 during retraction. The slow inhibition is mimicked and occluded by APGWamide, a neuropeptide contained in CBI-3. Thus, fast conventional and slow peptidergic transmissions originating from the same interneuron act in concert to meet specific temporal requirements in pattern switching.

Sonometric measurements of motor-neuron-evoked movements of an internal feeding structure (the radula) in Aplysia.

In many systems used to study rhythmic motor programs, the structures that generate behavior are at least partially internal. In these systems, it is often difficult to directly monitor neurally evoked movements. As a consequence, although motor programs are relatively well characterized, it is generally less clear how neural activity is translated into functional movements. This is the case for the feeding system of the mollusk Aplysia. Here we used sonomicrometry to monitor neurally evoked movements of the food-grasping organ in Aplysia, the radula. Movements were evoked by intracellular stimulation of motor neurons that innervate radula muscles that have been extensively studied in reduced preparations. Nevertheless our results indicate that the movements and neural control of the radula are more complex than has been assumed. We demonstrate that motor neurons previously characterized as radula openers (B48) and closers (B8, B15, B16) additionally produce other movements. Moreover, we show that the size of the movement evoked by a motor neuron can depend on the preexisting state of the radula. Specifically, the motor neurons B15 and B16 produce large closing movements when the radula is partially open but produce relatively weak closing movements in a preparation at rest. Thus the efficacy of B15 and B16 as radula closers is context dependent.

Cerebrin prohormone processing, distribution and action in Aplysia californica.

The isolation, characterization, and bioactivity in the feeding circuitry of a novel neuropeptide in the Aplysia californica central nervous system are reported. The 17-residue amidated peptide, NGGTADALYNLPDLEKIamide, has been termed cerebrin due to its primary location in the cerebral ganglion. Liquid chromatographic purification guided by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry allowed the isolation of the peptide with purity adequate for Edman sequencing. The cerebrin cDNA has been characterized and encodes an 86 amino acid prohormone that predicts cerebrin and one additional peptide. Mapping using in situ hybridization and immunocytochemistry showed that cerebrin containing neuronal somata are localized almost exclusively in the cerebral ganglion, mostly in the F- and C-clusters. Both immunostaining and mass spectrometry demonstrated the presence of cerebrin in the neurohemal region of the upper labial nerve. In addition, immunoreactive processes were detected in the neuropil of all of the ganglia, including the buccal ganglia, and in some interganglionic connectives, including the cerebral-buccal connective. This suggests that cerebrin may also function as a local signaling molecule. Cerebrin has a profound effect on the feeding motor pattern elicited by the command-like neuron CBI-2, dramatically shortening the duration of the radula protraction in a concentration-dependent manner, mimicking the motor-pattern alterations observed in food induced arousal states. These findings suggest that cerebrin may contribute to food-induced arousal in the animal. Cerebrin-like immunoreactivity is also present in Lymnaea stagnalis suggesting that cerebrin-like peptides may be widespread throughout gastropoda.

Targeting of peptidergic vesicles in cotransmitting terminals.

In the present study, we examined the targeting of neuropeptide-containing vesicles in terminals of neurons that release both neuropeptides and classical transmitters. Single neurons were electrically stimulated with patterns of activity that were recorded in freely behaving animals. The amount of peptide release was measured biochemically using a radioimmunoassay, and the targeting of peptidergic vesicles was quantified using immunoelectronmicroscopy. Repeated electrical stimulation of single neurons produced a very large increase in peptide release. Peptide release is paralleled by a twofold increase in the number of peptidergic vesicles docked at the portion of the terminal membrane that is away from the target muscle. This is in stark contrast to cholinergic vesicles, which aggregate at, and are released from the conventional release sites in close apposition to the muscle. This differential targeting of cholinergic and peptidergic vesicles may play a significant role in the distinct release requirements and spatial and temporal characteristics of the actions of conventional and peptidergic transmitters.

Cerebral-abdominal interganglionic coordinating neurons in Aplysia.

Three cerebral-abdominal interneurons (CAIs), CC2, CC3, and CC7, were identified in the cerebral ganglion C cluster. The cells send their axons to the abdominal ganglion via the pleural-abdominal connective. CC2 and CC3 are bilaterally symmetrical cells, whereas CC7 is a unilateral cell. CC3 is immunopositive for serotonin and may be the same cell (CB-1) previously described as located in the B cluster rather than the C cluster. We suggest that the full designation of CC3, be CC3(CB-1). All three cells respond to feeding-related inputs. Each CAI has a monosynaptic connection to at least one abdominal ganglion neuron involved in the control of various nonsomatic organs. The CAIs also exert widespread polysynaptic actions in the abdominal and head ganglia. The results suggest that the CAIs may act as interneurons that coordinate visceral responses mediated by the abdominal ganglion, with behaviors such as feeding and head withdrawal, that are controlled by neurons located in the head ganglia of the animal.

Cloning, expression and processing of the CP2 neuropeptide precursor of Aplysia.

The cDNA sequence encoding the CP2 neuropeptide precursor is identified and encodes a single copy of the neuropeptide that is flanked by appropriate processing sites. The distribution of the CP2 precursor mRNA is described and matches the CP2-like immunoreactivity described previously. Single cell RT-PCR independently confirms the presence of CP2 precursor mRNA in selected neurons. MALDI-TOF MS is used to identify additional peptides derived from the CP2 precursor in neuronal somata and nerves, suggesting that the CP2 precursor may give rise to additional bioactive neuropeptides.

Serotonergic and peptidergic modulation of the buccal mass protractor muscle (I2) in aplysia.

Plasticity of Aplysia feeding has largely been measured by noting changes in radula protraction. On the basis of previous work, it has been suggested that peripheral modulation may contribute to behavioral plasticity. However, peripheral plasticity has not been demonstrated in the neuromuscular systems that participate in radula protraction. Therefore in this study we investigated whether contractions of a major radula protraction muscle (I2) are subject to modulation. We demonstrate, first, that an increase in the firing frequency of the cholinergic I2 motoneurons will increase the amplitude of the resulting muscle contraction but will not modulate its relaxation rate. We show, second, that neuronal processes on the I2 muscle are immunoreactive to myomodulin (MM), RFamide, and serotonin (5-HT), but not to small cardioactive peptide (SCP) or buccalin. The I2 motoneurons B31, B32, B61, and B62 are not immunoreactive to RFamide, 5-HT, SCP, or buccalin. However, all four cells are MM immunoreactive and are capable of synthesizing MMa. Third, we show that the bioactivity of the different modulators is somewhat different; while the MMs (i.e., MMa and MMb) and 5-HT increase I2 muscle relaxation rate, and potentiate muscle contraction amplitude, MMa, at high concentrations, depresses muscle contractions. Fourth, our data suggest that cAMP at least partially mediates effects of modulators on contraction amplitude and relaxation rate.

Temporal pattern dependence of neuronal peptide transmitter release: models and experiments.

In this paper we construct, on the basis of existing experimental data, a mathematical model of firing-elicited release of peptide transmitters from motor neuron B15 in the accessory radula closer neuromuscular system of Aplysia. The model consists of a slow "mobilizing" reaction and the fast release reaction itself. Experimentally, however, it was possible to measure only the mean, heavily averaged release, lacking fast kinetic information. Considered in the conventional way, the data were insufficient to completely specify the details of the model, in particular the relative properties of the slow and the unobservable fast reaction. We illustrate here, with our model and with additional experiments, how to approach such a problem by considering another dimension of release, namely its pattern dependence. The mean release is sensitive to the temporal pattern of firing, even to pattern on time scales much faster than the time scale on which the release is averaged. The mean release varies with the time scale and magnitude of the pattern, relative to the time scale and nonlinearity of the release reactions with which the pattern interacts. The type and magnitude of pattern dependence, especially when correlated systematically over a range of patterns, can therefore yield information about the properties of the release reactions. Thus, temporal pattern can be used as a probe of the release process, even of its fast, directly unobservable components. More generally, the analysis provides insights into the possible ways in which such pattern dependence, widespread especially in neuropeptide- and hormone-releasing systems, might arise from the properties of the underlying cellular reactions.

Intrinsic and extrinsic modulation of a single central pattern generating circuit.

Intrinsic and extrinsic neuromodulation are both thought to be responsible for the flexibility of the neural circuits (central pattern generators) that control rhythmic behaviors. Because the two forms of modulation have been studied in different circuits, it has been difficult to compare them directly. We find that the central pattern generator for biting in Aplysia is modulated both extrinsically and intrinsically. Both forms of modulation increase the frequency of motor programs and shorten the duration of the protraction phase. Extrinsic modulation is mediated by the serotonergic metacerebral cell (MCC) neurons and is mimicked by application of serotonin. Intrinsic modulation is mediated by the cerebral peptide-2 (CP-2) containing CBI-2 interneurons and is mimicked by application of CP-2. Since the effects of CBI-2 and CP-2 occlude each other, the modulatory actions of CBI-2 may be mediated by CP-2 release. Although the effects of intrinsic and extrinsic modulation are similar, the neurons that mediate them are active predominantly at different times, suggesting a specialized role for each system. Metacerebral cell (MCC) activity predominates in the preparatory (appetitive) phase and thus precedes the activation of CBI-2 and biting motor programs. Once the CBI-2s are activated and the biting motor program is initiated, MCC activity declines precipitously. Hence extrinsic modulation prefacilitates biting, whereas intrinsic modulation occurs during biting. Since biting inhibits appetitive behavior, intrinsic modulation cannot be used to prefacilitate biting in the appetitive phase. Thus the sequential use of extrinsic and intrinsic modulation may provide a means for premodulation of biting without the concomitant disruption of appetitive behaviors.

Peptide profiling of cells with multiple gene products: combining immunochemistry and MALDI mass spectrometry with on-plate microextraction.

Due to the intracellular chemical complexity and a wide range of transmitter concentrations, the detection of the complete set of peptide transmitters in a single cell is problematic. In the current study, a multidisciplinary approach combining single-cell MALDI-MS peptide profiling, northern analysis, in situ hybridization, and immunocytochemistry allows characterization of a more complete set of neurotransmitters than individual approaches in the Aplysia californica B1 and B2 motor neurons. Because different results were obtained using both in situ and immunohistochemical techniques compared to previous reports, MALDI-MS assays have been used to examine CP1-related gene products in these cells. However, MALDI with standard sample preparation does not detect the presence of the CP1 gene products. A novel on-plate microextraction approach using concentrated MALDI matrix 2,5-dihydroxybenzoic acid with a mixture of acetone and water as the solvent has been developed to allow the detection of trace-level gene expression products. Both neuropeptide precursors in the B1 and B2 neurons-the SCP and CP1 prohormones-end with large peptides that have multiple cysteine residues. For SCP, MALDI-MS verifies the presence of a novel 9325 Da SCP-related peptide. In the case of CP1, a disulfide-bonded homodimer is detected and the disulfide bonding pattern elucidated using MALDI-MS coupled with on-plate enzymatic digestion.

Glutamate is the fast excitatory neurotransmitter of small cardioactive peptide-containing Aplysia radula mechanoafferent neuron B21.

B21 is a radula mechanoafferent neuron in the mollusc Aplysia which likely plays a crucial role in integrating environmental cues into the feeding motor program. To facilitate understanding B21's interactions with its postsynaptic followers, we sought to identify its neurotransmitter. We find that B21 makes a chemical synapse onto the follower neuron B8. Although B21-induced excitatory postsynaptic potentials (EPSPs) in B8 paradoxically diminish in amplitude with B8 hyperpolarization, we show that an inwardly rectifying current is responsible. We conclude that these B21-induced EPSPs are likely glutamatergic as they are blocked by the glutamate antagonist DNQX. Furthermore, B8 exhibits a depolarizing response to exogenous glutamate, which is antagonized by DNQX. Finally, exogenous glutamate occludes B21-evoked EPSPs in B8.