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Tissue-engineered tendon scaffolds have the potential to significantly improve the treatment of tendon and ligament injuries, especially those associated with tumors, trauma, and congenital deficiencies where autograft or allograft tissue might not be available in sufficient quantity for reconstruction. In this study, a tendon scaffold was produced that: (1) has decreased/absent cellular material histologically, as well as significantly decreased DNA content in comparison with the material it is derived from-fresh-frozen flexor digitorum profundus tendon; (2) is cytocompatible in vitro; (3) has been modified to produce increased pore size and porosity; (4) retains 76-78% of the tensile properties of the material it is derived from; (5) is readily infiltrated by fibroblast-like, mononuclear host cells; and (6) does not exhibit a host-cell-mediated foreign-body immune response after implantation in vivo.

The muscle and nerve responses to stresses applied during distraction osteogenesis have not been clearly defined. This study hypothesized that distraction resulting in 30% lengthening decreases muscle force generation of the lengthened muscle and increasing the frequency of distraction attenuates the decrease of force generation accompanying lengthening. This study investigated the effects of different distraction frequencies on neuromuscular recovery in a rabbit model. Animals were assigned into group 1 (low-frequency distraction) and group 2 (high-frequency distraction). Distraction was continued until a 30% increase in the original tibial length was achieved. After consolidation of the osteotomy, knee and ankle range of motion, muscle force generation, and neuromuscular junction parameters were evaluated. Lengthening of 30% resulted in significantly decreased range of motion compared with the control leg (P < 0.05). Lengthening of 30% also substantially decreased force generation of the peroneus longus muscle. However, force generation of the peroneus longus muscle in the high-frequency group was 70.5% +/- 6.5% of the control side, significantly higher than that in the low-frequency distraction group (49.7% +/- 4.8% of the control side, P < 0.05). There was no statistical difference between the 2 groups in neuromuscular junction morphology, although an abnormal shape of the postsynaptic neuromuscular junction was observed after distraction. The use of a high-frequency distraction technique during limb lengthening may result in a reduction in impairment of knee and ankle range of motion and improved muscle function compared with that observed with the use of low-frequency distraction. Repeated microtrauma to the soft tissues associated with high-frequency distraction may facilitate the regenerative capacity of the soft tissues and result in an improved outcome of muscle and nerve function.

Abnormal posttraumatic pain may delay recovery and severely impact health-related quality of life. The term complex regional pain syndrome describes abnormally intense and prolonged pain that is not related to tissue damage and is sometimes a sequela of injury. Various treatment strategies, including therapy, parental interventions, and peripheral surgery, are used to manage the condition.

Complex regional pain syndrome (CRPS) is a clinical syndrome of pain, autonomic dysfunction, trophic changes, and functional impairment. CRPS is common after hand trauma or surgery. Early diagnosis and intervention is critical for adequate recovery. The diagnosis of CRPS requires a careful history, physical examination, and supporting diagnostic testing. Optimal treatment requires a multidisciplinary approach. A large spectrum of pharmacologic interventions is efficacious in treating CRPS. Surgery may be used to relieve nociceptive foci. Patient-specific hand therapy is very important in reducing swelling, decreasing pain, and improving range of motion.

Purpose: Botulinum toxin A (BoNT-A) is used to manage spasticity in cerebral palsy. BoNT-A cleaves SNAP-25 protein, blocking acetylcholine release and weakening the muscle. Nicotinic acetylcholine receptors (nAChR) including alpha, beta, delta, gamma, and epsilon subunits, and GAP-43 protein are associated with functional recovery of neuromuscular junctions (NMJ) following BoNT-A. To better understand the mechanism behind this functional recovery, this study attempted to (1) document changes in NMJ morphometry following BoNT-A, and (2) determine the gene expression of nAChR subunits, SNAP-25, and GAP-43 protein. Methods: In this rat study (46 rats), 6 units/kg body weight of BoNT-A was injected into the gastrocnimus. NMJ morphometry and the time course of gene expression of nAChR subunits, SNAP-25, and GAP-43 were evaluated up to 1 year post-injection. Results: NMJ morphometry: gutter depth was reduced vs. the control side at two months, and normalizing by 6 months following BoNT. nAChR alpha mRNA and gamma mRNA increased by 3 days, peaked at 7 days and returned to control levels; delta mRNA peaked at 3 days. Epsilon mRNA peaked by 7 days. SNAP-25 mRNA increased from 60 to 90 days, returning to control levels by 6 months. GAP-43 mRNA was unchanged. Conclusions: Specific nAChR subunit mRNA expression up-regulates and then returns to normal within two weeks, preceding changes in NMJ morphometry. Although GAP-43 participates in nerve sprouting, no increase of GAP-43 mRNA occurred following BoNT-A. Delayed up-regulation of SNAP-25 mRNA might be associated with muscle functional recovery.

Controlled joint extension followed by gradual distraction with use of an external fixator may facilitate primary repair of peripheral nerve defects by permitting end-to-end repair without tension. The hypothesis of the present study was that gradual lengthening of nerve repairs with use of incremental distraction would provide superior results compared with grafting or repair under tension.A median nerve segment measuring four times the diameter of the nerve was resected in thirty-six rabbits to create a 7-mm gap in the nerve. Neurorrhaphy was performed with use of one of three techniques. In Group 1 (cable graft), a tension-free medial antebrachial cutaneous graft was placed to allow full range of motion of the elbow postoperatively. In Group 2 (end-to-end repair without distraction), the elbow was externally fixed in hyperflexion and the nerve was repaired end-to-end. At fourteen days, the fixator was removed and unprotected elbow motion was permitted. In Group 3 (end-to-end repair with gradual distraction), the elbow was externally fixed in hyperflexion and primary neurorrhaphy was performed. At fourteen days, the elbow was extended 10 degrees every other day with use of the articulated external fixator until full extension was achieved. Median nerve amplitude, latency, and nerve-conduction velocity; flexor digitorum superficialis single-twitch force generation and maximum tetanic force generation; muscle mass; and elbow range of motion were measured at three or six months. In addition, histologic analysis of the median nerve distal to the repair site and the morphometry of the neuromuscular junction in the flexor digitorum superficialis were performed at six months.All rabbits regained full active and passive range of motion. At three months, the nerve-conduction velocities in Groups 2 and 3 were significantly greater than that in Group 1. At six months, the nerve-conduction velocities and amplitudes in Group 3 were significantly greater than those in Groups 1 and 2. At six months, the tetanic force in Group 3 was significantly greater than those in Groups 1 and 2. There were no significant differences in muscle mass among the groups. There were no significant differences in histological findings among the three groups, although there was a trend toward larger fiber size in Group 3 as compared with the other two groups. The neuromuscular junctions in Group 3 had a significantly larger surface area than did those in Group 1 (p = 0.002) and Group 2 (p = 0.034).The use of an articulated external fixator and controlled gradual distraction appears to facilitate the treatment of peripheral nerve defects.

The aim of this study was to compare the systemic hemodynamic effects of four commonly used anesthetic regimens in mice that were chronically instrumented for direct and continuous measurements of cardiac output (CO). Mice (CD-1, Swiss, and C57BL6 strains) were instrumented with a transit-time flow probe placed around the ascending aorta for CO measurement. An arterial catheter was inserted into the aorta 4 or 5 days later for blood pressure measurements. After full recovery, hemodynamic parameters including stroke volume, heart rate, CO, mean arterial pressure (MAP), and total peripheral resistance were measured with animals in the conscious state. General anesthesia was then induced in these mice using isoflurane (Iso), urethane, pentobarbital sodium, or ketamine-xylazine (K-X). The doses and routes of administration of these agents were given as required for general surgical procedures in these animals. Compared with the values obtained for animals in the conscious resting state, MAP and CO decreased during all anesthetic interventions, and hemodynamic effects were smallest for Iso (MAP, −24 ± 3%; CO, −5 ± 7%; n = 15 mice) and greatest for K-X (MAP, −51 ± 6%; CO, −37 ± 9%; n = 8 mice), respectively. The hemodynamic effects of K-X were fully antagonized by administration of the α2-receptor antagonist atipamezole ( n = 8 mice). These results indicate that the anesthetic Iso has fewer systemic hemodynamic effects in mice than the nonvolatile anesthetics.

Cutaneous perfusion of the feet of normal volunteers and diabetic patients were measured by laser Doppler fluxmetry. A test paradigm was followed in which the feet were studied at room temperature, during cooling, and during rewarming after cooling. The data files were analyzed using sequential Fourier techniques on serial 20 second segments. Contour plots were generated from the resulting 120 Fourier spectra and compared between the diabetic patients and the normal volunteers. The use of contour plots was found to be helpful in differentiating the diabetic from normal foot plots during this type of stress testing. Significant portions of the power spectra were found in the low frequencies (0.1-0.2 Hz) but there was also a strong component corresponding to the cardiac cycle with at least one harmonic of that cycle observed at two times the heart rate. Diabetic patients exhibited reductions in cutaneous perfusion with cooling in both the low frequency and heart rate frequencies. Sequential spectral analyses arranged in contour plots provide significant information about cutaneous perfusion using laser Doppler fluxmetry and prove useful in evaluating other pathologies characterized by cutaneous perfusion abnormalities. >

Anatomical and physiological differences in neuromuscular junctions (NMJs) between juvenile and adult muscle may partially explain the variability in clinical results following chemodenervation with botulinum toxin or nerve repair in children and adults. We evaluated NMJ density, distribution, and morphometry in biceps brachii and gastrocnemius muscles from juvenile and adult rats. Motor endplates were stained with Karnovsky–Roots methods, and NMJ density (number/gram muscle tissue) was determined. The NMJ morphometry was quantitated with alpha-bungarotoxin labeling using confocal microscopy. Juvenile rats had a greater NMJ density in both muscles compared with adult rats. Juveniles and adult rats had a similar NMJ distribution in both muscles. In juvenile muscles NMJs occupied approximately 50% of the surface area and 70% of the length, width, circumference, and gutter depth compared with adult muscles. Our study demonstrates that although NMJs are smaller, juvenile muscles have a higher NMJ density than do adult muscles with similar distributions. If these age-dependent differences in NMJ density are obtained in humans, they may account, at least in part, for the better recovery that occurs in children than adults after nerve repair and also suggest that the dosage of botulinum toxin (units per kg) for chemodenervation may need to be increased in juveniles. © 2002 Wiley Periodicals, Inc. Muscle Nerve 26: 804–809, 2002

The use of periarterial sympathectomy (PAS) to manage chronic digital ischemia caused by scleroderma remains controversial. The duration of efficacy of PAS in managing scleroderma symptoms was evaluated by examining microvascular physiology, health-related quality of life, and patient satisfaction. Twenty-two patients (29 hands) with scleroderma, chronic vascular insufficiency, and a history of nonhealing digital ulcers unresponsive to nonsurgical treatment were evaluated. Isolated cold stress testing and laser Doppler fluxmetry measurements were used to evaluate microvascular perfusion before surgery and after a mean follow-up period of 31 months (range, 7-108 months). Digital temperatures did not change after surgery, but microvascular perfusion had increased significantly at a mean of 31 months in 22 hands. Health-related quality-of-life data were collected at a mean of 46 months (range, 11-108 months) after surgery. Subjective improvement (fewer ulcers, faster ulcer healing, and decreased pain) was reported for 18 of 22 patients (24 of 29 hands) at a mean follow-up period of 46 months; 6 patients remained ulcer free. Follow-up of scleroderma patients after PAS documented improved microvascular perfusion as evaluated by laser Doppler fluxmetry and by variable clinical results.

Reflex sympathetic dystrophy (RSD) is a complex pathologic entity that may occur following surgery or trauma, resulting in abnormally intense and inappropriately prolonged pain that is not a reflection of actual or impending tissue damage in an affected area. Until recently, the syndrome was not well described in the knee. Persistent knee pain was traditionally thought to be due to various posttraumatic or postoperative conditions.1 The classification of post-traumatic pain is complicated. RSD is well established in the lay, medical, and legal literature despite the absence of defined pathophysiology or consistent clinical symptoms or signs. The term reflex sympathetic dystrophy, however, may inappropriately imply that the sympathetic nervous system is actively involved without proof of increased sympathetic activity. Disorders considered to be RSD and causalgia are now classified under the term complex regional pain syndrome (CRPS) and are based entirely on clinical criteria. The new terminology of CRPS will be used throughout the text except where RSD was originally used by cited authors.

Neurokinin A (NKA) mediated a concentration dependent increase in the intracellular free Ca 2+ concentration, [Ca 2+ ] i , in B82 fibroblasts transfected with the neurokinin 2 (NK 2 ) receptor. The EC 50 value of this response was 24 nM. A selective NK 2 antagonist, MEN 10207, at a concentration of 1 μ M completely inhibited the [Ca 2+ ] i rise to 0.1 μ M NKA. These results suggest that activation of NK 2 receptors expressed in the transfected fibroblasts are functionally coupled to intracellular calcium mobilization.

The authors investigated the effects of closed soft tissue injury (CSTI) and cryotherapy on microvascular perfusion by means of laser Doppler flowmetry. In a different protocol interactions between leukocytes and the microvascular endothelium of skeletal muscle, which are first steps in local inflammatory response were documented in an intravital microscopy model. Rats were chronically instrumented with dorsal skinfold chambers. Leukocyte rolling and adherence in postcapillary venules of striated muscle before and after standardized muscle contusion (group T: n = 6), cryotherapy following muscle contusion (TK: n = 6) or sham contusion (group N: n = 6) were quantitated prospectively and in random order using intravital microscopy. Tissue perfusion was assessed by laser Doppler flowmetry in the microvasculature of the flap over a period of 96 hours following CSTI (group T: n = 8), cryotherapy following CSTI (group TK: n = 8), sham trauma (group N: n = 8, control 1), or cryotherapy following sham trauma (group K: n = 8, control 2). 300 minutes after trauma the number of rolling and adherent leukocytes in striated muscle microvasculature was significantly reduced by cryotherapy. We found an acute effect of cryotherapy on perfusion of traumatized tissue (reduction of perfusion around 25 %). However, there was no long-term effect (96 hours evaluation) on microvascular perfusion of cryotherapy either in the presence or absence of CSTI. The effectiveness of ice application in reducing edema in striated muscle following contusion may be due in part to a reduction of the leukocyte/endothelial interactions accompanying CSTI.

The use of mice as models for cardiovascular studies has traditionally been difficult because of their small size and the lack of appropriate instrumentation to perform fundamental measurements of cardiac output (CO) and total blood volume (TBV). The advent of transgenic techniques to develop mouse strains that mimic human disease makes the development of this instrumentation crucial. The current study outlines a novel technique for the determination of CO and TBV in the mouse using an extracorporeal arteriovenous (A-V) shunt, combined with the measurement of ultrasound dilution after the intravenous administration of small volumes of isotonic saline. The potential sources of error associated with Stewart-Hamilton dilution techniques were addressed by the research. The new techniques were applied in three anesthetized mice (27–36 gm). Isotonic saline (10–80 μl) was injected intravenously while measuring ultrasound dilution in the A-V shunt. The CO ranged from an average of 6.8 ± 0.71 to 12.7 ± 1.7 ml/min. Heart rates were not significantly altered by the intravenous administration of isotonic saline. The TBV ranged from 4.36 ± 0.22 to 5.15 ± 1.04 ml/100 gm. These results agree with the literature and suggest that these techniques will prove useful in cardiovascular studies of mice. ASAIO Journal 1999; 45:32–36.

OBJECTIVE To investigate the relationship between secondary muscle damage after contusion and the interactions between leukocytes and endothelial cells, which are essential steps in secondary inflammatory response. METHODS In a randomized animal study, rats were chronically instrumented with dorsal skinfold microvascular chambers and exposed to standardized contusion or sham contusion. Leukocyte rolling and adherence in postcapillary venules before and after muscle contusion or sham contusion were quantitated using in vivo microscopy. RESULTS The number of rolling leukocytes in the postcapillary venules before contusion was low. At 300 minutes after contusion, the number of rolling and adherent leukocytes in the striated muscle microvasculature was increased significantly (p < or = 0.05) compared with either the baseline precontusion condition or the control group at the same time. CONCLUSION In the mid-term to long-term stages of skeletal muscle injury associated with contusion, a significant portion of tissue damage is secondary to leukocyte-endothelial cell interactions.

Stem cells have become an important component of tissue regeneration, as they are able to differentiate into various cell types if guided appropriately. It is well known that cellular differentiation is greatly influenced by the surrounding microenvironment. We have developed a composite scaffold system using a collagen matrix derived from porcine bladder submucosa matrix (BSM) and poly(lactide-co-glycolide) (PLGA). In this study, we investigated whether a composite scaffold composed of naturally derived matrix combined with synthetic polymers would provide a microenvironment to facilitate the induction of osteogenic differentiation. We first showed that human amniotic fluid-derived stem cells (hAFSCs) adhered to the composite scaffolds and proliferated over time. We also showed that the composite scaffolds facilitated the differentiation of hAFSCs into an osteogenic lineage. The expression of osteogenic genes, including RUNX2, osteopontin (OPN) and osteocalcin (OCN) was upregulated in cells cultured on the composite scaffolds incubated in the osteogenic medium compared with ones without. Increased alkaline phosphatase (ALP) activity and calcium content indicates that hAFSCs seeded on 3D porous BSM–PLGA composite scaffolds resulted in higher mineralization rates as the duration of induction increased. This was also evidenced by the mineralized matrix within the scaffolds. The composite scaffold system provides a proper microenvironment that can facilitate osteogenic differentiation of AFSCs. This scaffold system may be a good candidate material for bone tissue engineering.

Before 1993, extant populations of Michaux S sumac (Rhus michauxil), a shrub federally listed as endangered, were known only from North Carolina and Georgia. In 1993, a large population of Rhus michauxii was found in the southern Piedmont of Virginia at the Fort Pickett Military Reservation in habitats subjected to ordnance- caused fires and soil disturbances within the Fort's Controlled Access Area (CAA). After the initial surveys in 1993, additional areas of Fort Pickett remained to be searched for Michaux's sumac, and in October 1994-March 1995 an extensive survey was conducted by the Virginia Department of Conservation and Recreation. Selected areas both inside and outside the CAA were searched. This survey resulted in the discovery of 74 colonies of Rhus michauxii, with 36 colonies containing 1,800+ stems outside of the CAA and 38 colonies containing 6,000+ stems within the CAA. Most of the colonies were found in habitats subjected to ordnance-caused disturbances, but some were found in disturbed habitats around old homesites, strengthening that association found previously by Fort Pickett personnel.

Nanotubes (NT) composed of the electrically active polymer poly (3,4-ethylenedioxythiophene) (PEDOT) have been used for photothermal ablation of both gram-positive (Staphylococcus aureus) and gram-negative (Escherichia coli) bacteria. Since infrared absorption of PEDOT is dominated by bipolarons strongly coupled to phonons, we hypothesize that nonradiative decay of these states leads to heat generation. Photothermal death of bacteria by PEDOT NT was compared to single-wall carbon nanotubes (SWNT). Complete eradication of bacterial colonies incubated with 100 ug/ml of either PEDOT NT or SWNT occurred with a single exposure to 1064 nm light (3.8 W/cm2) for 60 s. PEDOT NT were also shown to elicit a mild antibacterial response upon incubation with bacteria and no infrared exposure. PEDOT NT have the same capacity for photothermal ablation of bacteria as compared to SWNT; therefore, they represent an exciting new class of polymer based nanoparticles for medically-relevant photothermal therapies.

Conditions such as Raynaud's disease, cold intolerance following digit replantation, and reflex sympathetic dystrophy are all exacerbated by diminished blood flow in the digits. The rabbit ear, being similar to the human digit, is an excellent model for studying microvascular responses during thermoregulation and was used in this study of microcirculatory changes during total-body cold stress. In eight rabbits a window was placed in one ear each, and the underlying vascular beds were photographed with the unanesthetized animal at room temperature (23°C) and in a cold room (6°C). Luminal arterial diameters constricted more than venular diameters during cold stress (39.6% vs. 16.5%, respectively, of the control diameters; P = 0.014). All responses were linear; the slopes of the regression lines were significantly different between arterioles and venules (P < 0.0001). This study suggests that decreased circulation in response to cold is due to decreases in the diameter and number of vessels available for perfusion, as predicted by the Poiseuille equation. © 1994 Wiley-Liss, Inc.

The cellular injury produced by reperfusion of ischemic tissue with oxygen-rich blood has been studied in numerous tissues but has not been investigated extensively in thermoregulatory tissue. This study was designed (a) to compare 4 and 6 hours of ischemia to document the evidence of impaired capillary perfusion after resumption of blood flow (reperfusion injury) in a thermoregulatory end organ (the rabbit ear), and (b) to examine, with use of vital capillaroscopy (VC) and laser Doppler flowmetry (LDF), the altered microvascular blood flow in the rabbit ear after ischemia and reperfusion. One ear from each of five rabbits underwent warm ischemia for 4 hours. VC showed no deficits of capillary perfusion in these ears after reperfusion; LDF measurements in both ears also demonstrated no significant difference between control and reperfusion blood flow. One ear from each of eight additional rabbits underwent 6 hours of warm ischemia. LDF values were significantly reduced in the ischemic ear after reperfusion as compared with baseline measurements for that ear and as compared with the control ear. VC showed arrested perfusion and static plasma gaps within three to five capillaries per high-power field (an area of 300 x 500 microns) in the ischemic ear and good perfusion of all vessels in the contralateral control ear. This evidence of reperfusion injury in a thermoregulatory end organ may help to explain the poor functional result that often occurs after replantation of an amputated digit.

A rabbit ear model resembling the human digit was studied to determine the vascular response of the rabbit ear to a cold stress. Following moderate cooling (10 minutes at 5°–8°C), auricular blood flow and cutaneous perfusion were reduced. This decrease was reversed by 30 minutes of warming. The response in the rabbit ear to cold stress is similar to that of normal human digits. The similarities between the control of the circulation in human digits and rabbit ears may result from the similarities in digital and auricular vascular receptors and receptor subtypes. Verification of the rabbit model provides an experimental method for obtaining important data regarding digital pathophysiology and the treatment of cold intolerance. Further study with this model will provide clinically relevant information regarding the pathophysiology of digital thermoregulatory abnormalities. © 1994 Wiley-Liss, Inc.

Prostate cancer is the most frequently diagnosed malignancy and the second-leading cause of cancer death in men. We previously showed that angiotensin-(1-7) [Ang-(1-7)], a seven amino acid peptide hormone, significantly inhibited the growth of human lung cancer cells and tumors, with an associated reduction in angiogenesis. Since previous epidemiological studies suggest that administration of anti-hypertensive drugs which increase Ang-(1-7) reduces the risk of sex-specific cancers, we investigated the effects of the heptapeptide on prostate cancer. Ang-(1-7) markedly reduced human LNCaP prostate tumor xenograft size by 72% in association with a decrease in Ki67 and CD34, markers of tumor proliferation and angiogenesis, respectively. Ang-(1-7) significantly decreased both vascular endothelial growth factor (VEGF) and placental growth factor (PlGF) with a concomitant 12-fold increase in the soluble fraction of VEGF receptor 1 (sFlt-1); sFlt-1 is a decoy receptor that traps PlGF and VEGF, rendering the ligands unavailable to membrane-associated VEGF receptors. Ang-(1-7) also inhibits metastasis of prostate cancer to bone, which is the primary cause of mortality in prostate cancer patients. Human prostate cancer cells were injected into the circulation of SCID mice pretreated with Ang-(1-7), to determine the effect of the heptapeptide on the migration of cells to the metastatic environment. Six weeks following the injection of stably transfected luciferase tagged PC3 (PC3Luc) cells, 5 of the 6 untreated mice developed metastatic bone tumors, measured by bioluminescence and MRI imaging; in contrast, no detectable tumors were observed in mice administered Ang-(1-7). Circulating VEGF was significantly higher in untreated mice compared to mice treated with the heptapeptide. Ang-(1-7) also significantly reduced metastatic tumor formation in athymic mice injected with PC3Luc cells in the tibia as determined by bioluminescence, MRI imaging and immunohistochemistry. Osteolytic lesions as assessed by tartrate resistant acid phosphatase (TRAP) staining were observed surrounding the tibial tumors in control animals. A 50% reduction in osteoclastogenesis was observed when bone marrow cells were differentiated with RANK ligand and colony-stimulating factor in the presence of Ang-(1-7) [from 78.6 ± 8.0 TRAP+-multinucleated cells/field to 33.6 ± 4], suggesting that Ang-(1-7) hinders tumor survival in the bone microenvironment and prevents the formation of osteolytic lesions. Since VEGF is known to facilitate tumor growth and osteolytic disease by enhancing osteoclast survival, the inhibition of VEGF coupled with the reduction in osteoclastogenesis may mediate the inhibition of metastatic skeletal tumor formation. These results suggest that Ang-(1-7) may serve as an anti-proliferative, anti-angiogenic, and anti-metastatic agent for the treatment of prostate cancer that targets the tumor microenvironment. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 550. doi:10.1158/1538-7445.AM2011-550

After chronic exposure to neurokinin A, a time-dependent and recoverable desensitization of inositol monophosphate accumulation occurred in B82 fibroblasts transfected with cDNAs encoding for the bovine stomach NK2 receptor. Desensitized cells also showed decreased Ca2+ mobilization. While NK2 receptor antagonists had no effect on inositol monophosphate accumulation, substance P and senktide both produced a small degree of desensitization.

A new implantable window was developed in order to study the native microcirculation of the rabbit ear. The responses of the ear microvasculature to total body cooling were studied before and after implantation of the window. No differences were found between arteriolar and venular vasoconstriction with cooling before and after implantation of the window. Arteriolar constriction was significantly greater than the venular response both before and after placement of the window (P = 0.014). Surface skin temperature of the instrumented and control ears was similar at room temperature (35.2 +/- 2.6 versus 36.1 +/- 1.0 degrees, respectively; P = 0.280); however, the instrumented ear was slightly warmer during cooling (17.9 +/- 1.9 versus 15.2 +/- 3.1 degrees; P = 0.024), suggesting increased blood flow in the instrumented ear. Details of construction and implantation of the window are described.

Muscarinic receptor heterogeneity has been implicated in the selective binding characteristics of non-classical muscarinic antagonists such as pirenzepine (PZ) and AF-DX 116 (ll-[[2-[(diethyl-amino)methyl]-l-piperi-dinyl]acetyl]-5,ll-dihydro-6H-pyrido [2,3-b]-[l,4]benzodiazepine-6-one) (Watson et al., 1986). Pirenzepine labels a class of muscarinic receptor binding sites with high affinity, termed the Mi type, which is predominant in the brain. AF-DX 116, on the other hand, distinguishes a second class of binding sites by its high affinity, known as the M2 type, which is predominant in cardiac tissues (Giachetti et al., 1986). A third class of muscarinic receptor sites, noted for its low affinity for both PZ and AF-DX 116, is represented in a number of glandular tissues (Hammer et al., 1986; Korc et al., 1987). The heterogeneity of the muscarinic receptors was proven unequivocally by the identification of several genes (mi-m5, genotypic definition established by Bonner et al., 1987, 1988) which encode distinct polypeptides that show muscarinic cholinergic receptor properties (Bonner et al., 1987, 1988; Peralta et al., 1987; Liao et al., 1989). These structurally distinct types of the muscarinic receptors could be individually analyzed, by means of in vitro expression in eukaryotic cell lines, in order to correlate the structural diversity of the muscarinic receptors with their multiple physiological activities.

The chronic cranial window preparation allows repeated measurements of the same pial vessels in unanesthetized rats for several weeks and correlation with 24-h monitoring of hemodynamic variables. Nimodipine (20 mg) or placebo was given via two subcutaneous pellets. Large arterioles dilated 26 and 16%, at hour 1 and days 6-13, respectively (P less than 0.02). There was an increase in number of small arterioles throughout the whole observation period with the maximal increment of 47% (P less than 0.05) at days 6-13. Maximal vasodilation with 10% CO2 indicated that the increase in number of small arterioles after administering nimodipine was not caused by the opening of previously closed vessels. The total length of small arterioles and venules increased 47 and 23% at days 6-13, respectively (P less than 0.001). These increases seem to be caused by the increases in the numbers of vessels, because the average length of the small vessels did not appear to change. This suggests that nimodipine reduces cerebral vascular resistance by causing cerebral microvessel neovascularization. Our data demonstrate that the administration of nimodipine (20 mg) is potent in dilating pial arterioles in the short-term without affecting systemic arterial pressure, and that its long-term effect results in new vessel growth.

Objective: To evaluate the impact of complex regional pain syndrome (CRPS) after distal radius fractures on function and health-related quality of life (HRQOL). Design: Retrospective and prospective clinical study. Setting: University orthopedic department. Participants: 28 patients with CRPS after distal radius fractures. Interventions: Fracture management (closed reduction, cast immobilization, external fixation, open reduction and internal fixation, percutaneous pinning) and CRPS management (autonomic nerve blocks, oral medications, hand therapy, median nerve decompression). Main Outcome Measures: Clinical findings; thermoregulatory testing (isolated cold stress testing, laser Doppler measurements of cutaneous perfusion and digital pulp temperature); and HRQOL instruments (McGill Pain Questionnaire, McCabe Cold Sensitivity Scale, Levine Symptom Severity and Functional Scale, Disabilities of Arm, Shoulder, and Hand [DASH] questionnaires, Wake Forest University Symptom Rating Scale). All 3 measures were compared at initial presentation and at a mean follow-up of 5 years. Results: At 5-year follow-up, patients with CRPS after distal radius fractures who received the active intervention showed diminished pain, improved strength, and decreased stiffness; however, numbness and cold intolerance was not improved. Conclusion: CRPS after distal radius fractures results in a significantly diminished function, HRQOL, and residual disability despite active and vigorous intervention.

Objectives: To document the time course of recovery of the targeted muscle after botulinum toxin type A (BTX) injection. Design: Basic science study using a rat model. Setting: University medical center research laboratory. Animals: Twenty-four 1-month old Sprague-Dawley rats. Intervention: BTX-A was injected into the left gastrocnemius muscle of 24 rats (6U/kg of body weight). Equal volume of saline was injected into the right gastrocnemius muscle (control). Main Outcome Measures: Muscle mass, motor evoked action potentials (latency, amplitude), and muscle force generation (single twitch, tetanus) were evaluated at different follow-up intervals. Results: In the BTX group, muscle mass was reduced by 31.5% of the control side within 2 weeks of injection and recovered to 92% at 6 months. Latency was prolonged to 144.9% at 3 days and recovered to 101% at 6 months. Amplitude, single twitch, and tetanus were significantly reduced at 1 week, but recovered to >94% at 6 months. Conclusions: BTX injection at 6U/kg of body weight effectively decreased muscle mass and muscle electrophysiologic parameters with peak onset 1 to 2 weeks after injection and with >90% functional recovery at 6 months.

P193 Angiotensin-(1-7) [Ang-(1-7)] produces arteriolar dilation and a hypotensive action, which may be mediated through nitric oxide, prostaglandin or bradykinin mechanisms. In rats, Ang-(1-7) produces a biphasic pressor/depressor action; the pressor component is blocked by AT 1 antagonists while the depressor component is not blocked by either AT 1 or AT 2 antagonists. In this study, we determined whether Ang-(1-7) would produce cardiovascular effects in AT 1a receptor knockout mice. Male Agtr1a(-/-) (n = 5) and control mice (n = 6) were anesthetized with ketamine:xylazine (71:7 mg/kg) and received femoral artery and jugular vein catheters. As previously reported, baseline blood pressure was lower (62 ± 5 mm Hg) in the Agtr1a(-/-) than in the controls (79 ± 6 mm Hg). Arterial blood pressure responses to Ang-(1-7) (0.15 - 1.5 μmol/kg) were determined. Ang-(1-7) produced a dose dependent pressor response in control animals with the peak response at the highest dose (15 ± 2 mm Hg). The pressor response was followed immediately by a depressor response ranging from -6 ± 1 at the lowest dose to -8 ± 1 mm Hg for the highest dose. AT 1a receptor knockout mice showed no pressor response to Ang-(1-7) but did show an immediate depressor response ranging from -4 ± 1 to -6 ± 2 mm Hg. Similar reductions in blood pressure without pressor effects were observed in female knockout mice (-5 ± 2 mm Hg to -9 ± 2 mm Hg over the same dose range, n = 3). The results of this study indicate that the depressor response to Ang-(1-7) is not dependent on a preceding pressor response to this peptide. Furthermore, the depressor response is not mediated by an AT 1a receptor, consistent with our previous reports of a non-AT 1 , non-AT 2 receptor mediating the actions of Ang-(1-7) in the vasculature. (Support: HL51952; HL55082)

An acute in vivo preparation of the microvasculature of the rabbit ear was used to evaluate the functional role of alpha1 (alpha1)-adrenoceptor subtypes in thermoregulatory microcirculation. The effect of alpha1-adrenoceptor subtype blockade on phenylephrine-induced vasoconstriction was assessed with the alpha1A, alpha1B, and alpha1D-adrenoceptor-selective antagonists 5-methyl-urapidil (10(-8) M), chloroethylclonidine (10(-5) M), and 8-[2-[4(2-methoxyphenyl)-1-piperazinyl]ethyl]-8-azaspirol[4.5]deca ne-7,9-dione dihydrochloride (BMY7378) (10(-6) M), respectively. The results demonstrated that pretreatment of the ear microvasculature with 5-methyl-urapidil or BMY7378 shifted the phenylephrine concentration-response curve rightward and significantly changed the log of the phenylephrine concentration, causing half-maximum stimulation (EC50) in arterioles (p < 0.05). BMY7378 shifted the phenylephrine concentration-response curve of the arteriovenous anastomoses about 100-fold rightward (p < 0.05). All three alpha1-adrenoceptor antagonists eliminated the vasoconstrictive effects of phenylephrine on venules. The results indicate that the ear microvasculature has a heterogenous distribution of alpha1-adrenoceptor subtypes. The alpha1A and alpha1D-adrenoceptor subtypes appear to have a greater influence on constrictive function in arterioles, whereas the alpha1D-adrenoceptor is the dominant constrictor of arteriovenous anastomoses. In general, the alpha1-adrenoceptor does not play a major vasoconstrictor role in venules. Chloroethylclonidine, an irreversible alpha1B-adrenoceptor antagonist, induced contractile responses in the ear microvasculature, probably due to its alpha2-adrenoceptor agonist effects. This study extended our understanding of the adrenergic receptor control mechanisms of a cutaneous thermoregulatory end organ characterized by two parallel perfusion circuits providing nutritional and thermoregulatory functions.