Administration of TGF-ß Inhibitor Mitigates Radiation-induced Fibrosis in a Mouse Model.

BACKGROUND: Radiation-induced fibrosis is a long-term adverse effect of external beam radiation therapy for cancer treatment that can cause pain, loss of function, and decreased quality of life. Transforming growth factor beta (TGF-ß) is believed to be critical to the development of radiation-induced fibrosis, and TGF-ß inhibition decreases the development of fibrosis. However, no treatment exists to prevent radiation-induced fibrosis. Therefore, we aimed to mitigate the development of radiation-induced fibrosis in a mouse model by inhibiting TGF-ß. QUESTION/PURPOSES: Does TGF-ß inhibition decrease the development of muscle fibrosis induced by external beam radiation in a mouse model? METHODS: Twenty-eight 12-week-old male C57BL/6 mice were assigned randomly to three groups: irradiated mice treated with TGF-ßi, irradiated mice treated with placebo, and control mice that received neither irradiation nor treatment. The irradiated mice received one 50-Gy fraction of radiation to the right hindlimb before treatment initiation. Mice treated with TGF-c (n = 10) received daily intraperitoneal injections of a small-molecule inhibitor of TGF-ß (1 mg/kg) in a dimethyl sulfoxide vehicle for 8 weeks (seven survived to histologic analysis). Mice treated with placebo (n = 10) received daily intraperitoneal injections of only a dimethyl sulfoxide vehicle for 8 weeks (10 survived to histologic analysis). Control mice (n = 8) received neither radiation nor TGF-ß treatment. Control mice were euthanized at 3 months because they were not expected to exhibit any changes related to treatment. Mice in the two treatment groups were euthanized 9 months after radiation, and the quadriceps of each thigh was sampled. Masson's trichome stain was used to assess muscle fibrosis. Slides were viewed at 10 × magnification using bright-field microscopy, and in a blinded fashion, five representative images per mouse were used to quantify fibrosis. The mean ± SD fibrosis pixel densities in the TGF-ßi and radiation-only groups were compared using Mann-Whitney U tests. The ratio of fibrosis to muscle was calculated using the mean fibrosis per slide in the TGF-ßi group to standardize measurements. Alpha was set at 0.05. RESULTS: The mean (± SD) percentage of fibrosis per slide was greater in the radiation-only group (1.2% ± 0.42%) than in the TGF-ßi group (0.14% ± 0.09%) (odds ratio 0.12 [95% CI 0.07 to 0.20]; p < 0.001). Among control mice, mean fibrosis was 0.05% ± 0.02% per slide. Mice in the radiation-only group had 9.1 times the density of fibrosis as did mice in the TGF-ßi group. CONCLUSION: Our study provides preliminary evidence that the fibrosis associated with radiation therapy to a quadriceps muscle can be reduced by treatment with a TGF-ß inhibitor in a mouse model. CLINICAL RELEVANCE: If these observations are substantiated by further investigation into the role of TGF-ß inhibition on the development of radiation-induced fibrosis in larger animal models and humans, our results may aid in the development of novel therapies to mitigate this complication of radiation treatment.

Molecular and Metabolic Mechanism of Low-Intensity Pulsed Ultrasound Improving Muscle Atrophy in Hindlimb Unloading Rats.

Low-intensity pulsed ultrasound (LIPUS) has been proved to promote the proliferation of myoblast C2C12. However, whether LIPUS can effectively prevent muscle atrophy has not been clarified, and if so, what is the possible mechanism. The aim of this study is to evaluate the effects of LIPUS on muscle atrophy in hindlimb unloading rats, and explore the mechanisms. The rats were randomly divided into four groups: normal control group (NC), hindlimb unloading group (UL), hindlimb unloading plus 30 mW/cm2 LIPUS irradiation group (UL + 30 mW/cm2), hindlimb unloading plus 80 mW/cm2 LIPUS irradiation group (UL + 80 mW/cm2). The tails of rats in hindlimb unloading group were suspended for 28 days. The rats in the LIPUS treated group were simultaneously irradiated with LIPUS on gastrocnemius muscle in both lower legs at the sound intensity of 30 mW/cm2 or 80 mW/cm2 for 20 min/d for 28 days. C2C12 cells were exposed to LIPUS at 30 or 80 mW/cm2 for 5 days. The results showed that LIPUS significantly promoted the proliferation and differentiation of myoblast C2C12, and prevented the decrease of cross-sectional area of muscle fiber and gastrocnemius mass in hindlimb unloading rats. LIPUS also significantly down regulated the expression of MSTN and its receptors ActRIIB, and up-regulated the expression of Akt and mTOR in gastrocnemius muscle of hindlimb unloading rats. In addition, three metabolic pathways (phenylalanine, tyrosine and tryptophan biosynthesis; alanine, aspartate and glutamate metabolism; glycine, serine and threonine metabolism) were selected as important metabolic pathways for hindlimb unloading effect. However, LIPUS promoted the stability of alanine, aspartate and glutamate metabolism pathway. These results suggest that the key mechanism of LIPUS in preventing muscle atrophy induced by hindlimb unloading may be related to promoting protein synthesis through MSTN/Akt/mTOR signaling pathway and stabilizing alanine, aspartate and glutamate metabolism.

Pulsed electromagnetic field improves subchondral bone microstructure in knee osteoarthritis rats through a Wnt/ß-catenin signaling-associated mechanism.

Pulsed electromagnetic field (PEMF) is often used for management of osteoarthritis (OA). The aim of the study was to determine whether PEMF can successfully improve subchondral bone microstructure through a Wnt/ß-catenin signaling-associated pathway in rats with knee OA induced by low-dose monosodium iodoacetate (MIA). Seventy-two 12-week-old male Sprague-Dawley rats were randomly assigned to three groups: OA (n = 24), PEMF (n = 24), and Control (n = 24). OA was induced (OA and PEMF groups) by injecting 0.2 mg MIA in rats' right knee joint. The control rats received a single sterile saline injection in the right knee. Rats in the PEMF group were exposed to daily 2 h PEMF exposure with 75 Hz, 1.6 mT for 4 weeks. After 4 weeks, micro-computed tomography (micro-CT), real-time PCR, and immunohistochemistry staining were performed. The PEMF group increased bone volume fraction (BV/TV), trabecular thickness (Tb.Th), trabecular number (Tb.N), and suppressed bone surface/bone volume (BS/BV) and trabecular separation (Tb.Sp) levels in micro-CT analysis. Real-time PCR analysis showed that PEMF promoted tibial subchondral bone's gene expressions of Wnt3a, ß-catenin, and OPG, but did not alter LRP5 and RANKL mRNA levels. Similar results involved tibial subchondral bone's protein expressions that were observed in immunohistochemistry staining. These results suggest that PEMF preserved the structural integrity of subchondral bone in knee OA rats by promoting the activation of Wnt/ß-catenin signaling and OPG/RANKL/RANK signaling. Bioelectromagnetics. 39:89-97, 2018. © 2017 Wiley Periodicals, Inc.

Pulsed electromagnetic fields prevented the decrease of bone formation in hindlimb-suspended rats by activating sAC/cAMP/PKA/CREB signaling pathway.

Microgravity is one of the main threats to the health of astronauts. Pulsed electromagnetic fields (PEMFs) have been considered as one of the potential countermeasures for bone loss induced by space flight. However, the optimal therapeutic parameters of PEMFs have not been obtained and the action mechanism is still largely unknown. In this study, a set of optimal therapeutic parameters for PEMFs (50 Hz, 0.6 mT 50% duty cycle and 90 min/day) selected based on high-throughput screening with cultured osteoblasts was used to prevent bone loss in rats induced by hindlimb suspension, a commonly accepted animal model to simulate the space environment. It was found that hindlimb suspension for 4 weeks led to significant decreases in femoral and vertebral bone mineral density (BMD) and their maximal loads, severe deterioration in bone micro-structure, and decreases in levels of bone formation markers and increases in bone resorption markers. PEMF treatment prevented about 50% of the decreased BMD and maximal loads, preserved the microstructure of cancellous bone and thickness of cortical bone, and inhibited decreases in bone formation markers. Histological analyses revealed that PEMFs significantly alleviated the reduction in osteoblast number and inhibited the increase in adipocyte number in the bone marrow. PEMFs also blocked decreases in serum levels of parathyroid hormone and its downstream signal molecule cAMP, and maintained the phosphorylation levels of protein kinase A (PKA) and cAMP response element-binding protein (CREB). The expression level of soluble adenylyl cyclases (sAC) was also maintained. It therefore can be concluded that PEMFs partially prevented the bone loss induced by weightless environment by maintaining bone formation through signaling of the sAC/cAMP/PKA/CREB pathway. Bioelectromagnetics. 39:569-584, 2018. © 2018 Wiley Periodicals, Inc.

Identification and characterization of an injury-induced skeletal progenitor.

The postnatal skeleton undergoes growth, remodeling, and repair. We hypothesized that skeletal progenitor cells active during these disparate phases are genetically and phenotypically distinct. We identified a highly potent regenerative cell type that we term the fracture-induced bone, cartilage, stromal progenitor (f-BCSP) in the fracture callus of adult mice. The f-BCSP possesses significantly enhanced skeletogenic potential compared with BCSPs harvested from uninjured bone. It also recapitulates many gene expression patterns involved in perinatal skeletogenesis. Our results indicate that the skeletal progenitor population is functionally stratified, containing distinct subsets responsible for growth, regeneration, and repair. Furthermore, our findings suggest that injury-induced changes to the skeletal stem and progenitor microenvironments could activate these cells and enhance their regenerative potential.

Remote activation of biomolecules in deep tissues using near-infrared-to-UV upconversion nanotransducers.

Controlled activation or release of biomolecules is very crucial in various biological applications. Controlling the activity of biomolecules have been attempted by various means and controlling the activity by light has gained popularity in the past decade. The major hurdle in this process is that photoactivable compounds mostly respond to UV radiation and not to visible or near-infrared (NIR) light. The use of UV irradiation is limited by its toxicity and very low tissue penetration power. In this study, we report the exploitation of the potential of NIR-to-UV upconversion nanoparticles (UCNs), which act as nanotransducers to absorb NIR light having high tissue penetration power and negligible phototoxicity and emit UV light locally, for photoactivation of caged compounds and, in particular, used for photo-controlled gene expression. Both activation and knockdown of GFP was performed in both solution and cells, and patterned activation of GFP was achieved successfully by using upconverted UV light produced by NIR-to-UV UCNs. In-depth photoactivation through tissue phantoms and in vivo activation of caged nucleic acids were also accomplished. The success of this methodology has defined a unique level in the field of photo-controlled activation and delivery of molecules.

Expression of mPGES-1 and IP mRNA is reduced by LLLT in both subplantar and brain tissues in the model of peripheral inflammation induced by carrageenan.

The increase in PGE2 production by microsomal PGE synthase-1 (mPGES-1) in CNS contributes to the severity of the inflammatory and pain responses in the model of edema formation and hyperalgesia induced by carrageenan. PGI2, alike to PGE2, plays an important role in the inflammation. Low-level laser therapy (LLLT) has been used in the treatment of inflammatory pathologies, reducing both pain and the acute inflammatory process. In this work, we studied the effect of LLLT on the expression of both mPGES-1 and IP messenger RNA (mRNA), in either subplantar or total brain tissues obtained from rats submitted to model of edema formation and hyperalgesia induced by carrageenan administration. The test sample consisted of 30 rats divided into five groups: A1 (control-saline), A2 (carrageenan-0.5 mg/paw), A3 (carrageenan-0.5 mg/paw + LLLT), A4 (carrageenan-1.0 mg/paw), and A5 (carrageenan-1.0 mg/paw + LLLT). The animals from groups A3 and A5 were irradiated 1 h after induction of inflammation by carrageenan injection. Continuous-wave red laser with wavelengths of 660 nm and dose of 7.5 J/cm(2) was used. Six hours after carrageenan-induced inflammation, mPGES-1 and prostacyclin receptor (IP) mRNA expression were significantly increased both in subplantar and brain tissues. LLLT was able to reduce both mPGES-1 and IP mRNA expression in subplantar and brain tissues. We suggest that LLLT is able to reduce both inflammation and hyperalgesia observed in the model of edema formation and hyperalgesia induced by carrageenan, by a mechanism involving the decrease in the expression of both mPGES-1 and IP.

Proton FLASH: Impact of Dose Rate and Split Dose on Acute Skin Toxicity in a Murine Model.

PURPOSE: Preclinical studies have shown a preferential normal tissue sparing effect of FLASH radiation therapy with ultra-high dose rates. The aim of the present study was to use a murine model of acute skin toxicity to investigate the biologic effect of varying dose rates, time structure, and introducing pauses in the dose delivery. METHODS AND MATERIALS: The right hind limbs of nonanaesthetized mice were irradiated in the entrance plateau of a pencil beam scanning proton beam with 39.3 Gy. Experiment 1 was with varying field dose rates (0.7-80 Gy/s) without repainting, experiment 2 was with varying field dose rates (0.37-80 Gy/s) with repainting, and in experiment 3, the dose was split into 2, 3, 4, or 6 identical deliveries with 2-minute pauses. In total, 320 mice were included, with 6 to 25 mice per group. The endpoints were skin toxicity of different levels up to 25 days after irradiation. RESULTS: The dose rate50, which is the dose rate to induce a response in 50% of the animals, depended on the level of skin toxicity, with the higher toxicity levels displaying a FLASH effect at 0.7-2 Gy/s. Repainting resulted in higher toxicity for the same field dose rate. Splitting the dose into 2 deliveries reduced the FLASH effect, and for 3 or more deliveries, the FLASH effect was almost abolished for lower grades of toxicity. CONCLUSIONS: The dose rate that induced a FLASH effect varied for different skin toxicity levels, which are characterized by a differing degree of sensitivity to radiation dosage. Conclusions on a threshold for the dose rate needed to obtain a FLASH effect can therefore be influenced by the dose sensitivity of the used endpoint. Splitting the total dose into more deliveries compromised the FLASH effect. This can have an impact for fractionation as well as for regions where 2 or more FLASH fields overlap within the same treatment session.

Oxygen Enhancement Ratio-Weighted Dose Quantitatively Describes Acute Skin Toxicity Variations in Mice After Pencil Beam Scanning Proton FLASH Irradiation With Changing Doses and Time Structures.

PURPOSE: The aim of this work was to investigate the ability of a biological oxygen enhancement ratio-weighted dose, DOER, to describe acute skin toxicity variations observed in mice after proton pencil beam scanning irradiations with changing doses and beam time structures. METHODS AND MATERIALS: In five independent experiments, the right hind leg of a total of 621 CDF1 mice was irradiated previously in the entrance plateau of a pencil beam scanning proton beam. The incidence of acute skin toxicity (of level 1.5-2.0-2.5-3.0-3.5) was scored for 47 different mouse groups that mapped toxicity as function of dose for conventional and FLASH dose rate, toxicity as function of field dose rate with and without repainting, and toxicity when splitting the treatment into 1 to 6 identical deliveries separated by 2 minutes. DOER was calculated for all mouse groups using a simple oxygen kinetics model to describe oxygen depletion. The three independent model parameters (oxygen-depletion rate, oxygen-recovery rate, oxygen level without irradiation) were fitted to the experimental data. The ability of DOER to describe the toxicity variations across all experiments was investigated by comparing DOER-response curves across the five independent experiments. RESULTS: After conversion from the independent variable tested in each experiment to DOER, all five experiments had similar MDDOER50 (DOER giving 50% toxicity incidence) with standard deviations of 0.45 - 1.6 Gy for the five toxicity levels. DOER could thus describe the observed toxicity variations across all experiments. CONCLUSIONS: DOER described the varying FLASH-sparing effect observed for a wide range of conditions. Calculation of DOER for other irradiation conditions can quantitatively estimate the FLASH-sparing effect for arbitrary irradiations for the investigated murine model. With appropriate fitting parameters DOER also may be able to describe FLASH effect variations with dose and dose rate for other assays and endpoints.

Alteration of the inflammatory molecule network after irradiation of soft tissue.

Inflammatory molecules (IMs) play an important role in ionizing radiation (IR)-induced soft tissue damage. The alteration of IMs as a function of time was studied with a protein array containing 62 IMs in mouse cutaneous soft tissues exposed to 30 Gy. The results showed that: (1) 2 days after irradiation, the levels of TGF-ß1, MIP-1γ, IL-1α, and sTNF RI increased, while IGFBP-3, CXCL16, and IL-1ß decreased in IR skin as compared to control skin; (2) 21 days after IR, TGF-ß1, and MIP-1 γ, IL-1α remained high, while CXCL16 and IL-1ß remained low; (3) 3 months after IR, the cytokine pattern exhibited reversals. The levels of MIP-1γ decreased, while VCAM-1, IGFBP-3, and TGF-ß1 production increased. The data indicated that: (a) IMs change as a function of time after soft tissue irradiation; (b) changing IM levels may reflect the altered balance of the cytokine network, leading to imbalance or homeostasis; and (c) an antibody-based protein array can be used to assess multiple IMs simultaneously, making it useful for bulk screening for changes in tissue cytokine levels.

Pulsed electromagnetic fields stimulation affects BMD and local factor production of rats with disuse osteoporosis.

Pulsed electromagnetic fields (PEMF) have been used widely to treat nonunion fractures and related problems in bone healing, as a biological and physical method. With the use of Helmholtz coils and PEMF stimulators to generate uniform time-varying electromagnetic fields, the effects of extremely low frequency electromagnetic fields on bone mineral density (BMD) and local factor production in disuse osteoporosis (DOP) rats were investigated. Eighty 4-month-old female Sprague Dawley (SD) rats were randomly divided into intact (INT) group, DOP group, calcitonin-treated (CT) group, and PEMF stimulation group. The right hindlimbs of all the rats were immobilized by tibia-tail fixation except for those rats in the INT group. Rats in the CT group were injected with calcitonin (2 IU/kg, i.p., once a day) and rats in the PEMF group were irradiated with PEMF immediately postoperative. The BMD, serum transforming growth factor-beta 1 (TGF-beta1), and interleukin-6 (IL-6) concentration of the proximal femur were measured 1, 2, 4, and 8 weeks after treatment. Compared with the CT and DOP groups, the BMD and serum TGF-beta1 concentration in the PEMF group increased significantly after 8 weeks. The IL-6 concentration in the DOP group was elevated significantly after operation. The PEMF group showed significantly lower IL-6 level than the DOP group. The results found demonstrate that PEMF stimulation can efficiently suppress bone mass loss. We, therefore, conclude that PEMF may affect bone remodeling process through promoting TGF-beta1 secretion and inhibiting IL-6 expression.

Millimeter wave effects on electrical responses of the sural nerve in vivo.

Millimeter wave (MMW, 42.25 GHz)-induced changes in electrical activity of the murine sural nerve were studied in vivo using external electrode recordings. MMW were applied to the receptive field of the sural nerve in the hind paw. We found two types of responses of the sural nerve to MMW exposure. First, MMW exposure at the incident power density >/=45 mW/cm(2) inhibited the spontaneous electrical activity. Exposure with lower intensities (10-30 mW/cm(2)) produced no detectable changes in the firing rate. Second, the nerve responded to the cessation of MMW exposure with a transient increase in the firing rate. The effect lasted 20-40 s. The threshold intensity for this effect was 160 mW/cm(2). Radiant heat exposure reproduced only the inhibitory effect of MMW but not the transient excitatory response. Depletion of mast cells by compound 48/80 eliminated the transient response of the nerve. It was suggested that the cold sensitive fibers were responsible for the inhibitory effect of MMW and radiant heat exposures. However, the receptors and mechanisms involved in inducing the transient response to MMW exposure are not clear. The hypothesis of mast cell involvement was discussed.

Regional and Conducted Vascular Effects of Endovascular Ultrasound Catheters.

Intra-vascular ultrasound catheters are used clinically to facilitate clot lysis. We hypothesized that these devices could also directly lower microvascular resistance and increase tissue perfusion through established shear-dependent pathways. In mice, either the proximal hind-limb muscles or the upstream femoral artery alone was exposed to an endovascular ultrasound catheter (2.3 MHz, 0.5-1.1 MPa) for 10 min. Quantitative microvascular perfusion imaging in the hind limbs exposed to the endovascular ultrasound system exhibited a more-than-twofold increase in flow (p < 0.01) compared with the contralateral control limb after exposure of either the muscle or the femoral artery alone. Using an in vivo optical imaging reporting system, an eight- to ninefold increase in tissue adenosine triphosphate (ATP) was detected in the region of insonification (p = 0.006). Ultrasound was found to produce an immediate release of ATP from ex vivo erythrocytes (p = 0.03). In situ electrochemical sensing revealed an immediate increase in nitric oxide with initiation of ultrasound which returned to baseline within 5 min of termination, as well as ultrasound-triggered nitric oxide (NO) release from erythrocytes. These data indicate that non-cavitating ultrasound produced by endovascular catheters can reduce vascular resistance and increase flow through recognized shear-dependent vasodilator pathways involving purinergic signaling and NO.

Chemoradiation impairs myofiber hypertrophic growth in a pediatric tumor model.

Pediatric cancer treatment often involves chemotherapy and radiation, where off-target effects can include skeletal muscle decline. The effect of such treatments on juvenile skeletal muscle growth has yet to be investigated. We employed a small animal irradiator to administer fractionated hindlimb irradiation to juvenile mice bearing implanted rhabdomyosarcoma (RMS) tumors. Hindlimb-targeted irradiation (3 × 8.2 Gy) of 4-week-old mice successfully eliminated RMS tumors implanted one week prior. After establishment of this preclinical model, a cohort of tumor-bearing mice were injected with the chemotherapeutic drug, vincristine, alone or in combination with fractionated irradiation (5 × 4.8 Gy). Single myofiber analysis of fast-contracting extensor digitorum longus (EDL) and slow-contracting soleus (SOL) muscles was conducted 3 weeks post-treatment. Although a reduction in myofiber size was apparent, EDL and SOL myonuclear number were differentially affected by juvenile irradiation and/or vincristine treatment. In contrast, a decrease in myonuclear domain (myofiber volume/myonucleus) was observed regardless of muscle or treatment. Thus, inhibition of myofiber hypertrophic growth is a consistent feature of pediatric cancer treatment.

Thrombospondin-1 and CD47 limit cell and tissue survival of radiation injury.

Radiation, a primary mode of cancer therapy, acutely damages cellular macromolecules and DNA and elicits stress responses that lead to cell death. The known cytoprotective activity of nitric oxide (NO) is blocked by thrombospondin-1, a potent antagonist of NO/cGMP signaling in ischemic soft tissues, suggesting that thrombospondin-1 signaling via its receptor CD47 could correspondingly increase radiosensitivity. We show here that soft tissues in thrombospondin-1-null mice are remarkably resistant to radiation injury. Twelve hours after 25-Gy hindlimb irradiation, thrombospondin-1-null mice showed significantly less cell death in both muscle and bone marrow. Two months after irradiation, skin and muscle units in null mice showed minimal histological evidence of radiation injury and near full retention of mitochondrial function. Additionally, both tissue perfusion and acute vascular responses to NO were preserved in irradiated thrombospondin-1-null hindlimbs. The role of thrombospondin-1 in radiosensitization is specific because thrombospondin-2-null mice were not protected. However, mice lacking CD47 showed radioresistance similar to thrombospondin-1-null mice. Both thrombospondin-1- and CD47-dependent radiosensitization is cell autonomous because vascular cells isolated from the respective null mice showed dramatically increased survival and improved proliferative capacity after irradiation in vitro. Therefore, thrombospondin-1/CD47 antagonists may have selective radioprotective activity for normal tissues.

Timing in the absence of supraspinal input I: variable, but not fixed, spaced stimulation of the sciatic nerve undermines spinally-mediated instrumental learning.

Rats with complete spinal transections are capable of acquiring a simple instrumentally trained response. If rats receive shock to one hind limb when the limb is extended (controllable shock), the spinal cord will learn to hold the leg in a flexed position that minimizes shock exposure. If shock is delivered irrespective of leg position, subjects do not exhibit an increase in flexion duration and subsequently fail to learn when tested with controllable shock (learning deficit). Just 6 min of variable intermittent shock produces a learning deficit that lasts 24 h. Evidence suggests that the neural mechanisms underlying the learning deficit may be related to those involved in other instances of spinal plasticity (e.g. windup, long-term potentiation). The present paper begins to explore these relations by demonstrating that direct stimulation of the sciatic nerve also impairs instrumental learning. Six minutes of electrical stimulation (mono- or biphasic direct current [DC]) of the sciatic nerve in spinally transected rats produced a voltage-dependent learning deficit that persisted for 24 h (experiments 1-2) and was dependent on C-fiber activation (experiment 7). Exposure to continuous stimulation did not produce a deficit, but intermittent burst or single pulse (as short as 0.1 ms) stimulation (delivered at a frequency of 0.5 Hz) did, irrespective of the pattern (fixed or variable) of stimulus delivery (experiments 3-6, 8). When the duration of stimulation was extended from 6 to 30 min, a surprising result emerged; shocks applied in a random (variable) fashion impaired subsequent learning whereas shocks given in a regular pattern (fixed spacing) did not (experiments 9-10). The results imply that spinal neurons are sensitive to temporal relations and that stimulation at regular intervals can have a restorative effect.

Electrically evoked locomotor activity in the turtle spinal cord hemi-enlargement preparation.

We assessed the locomotor capacity of the left half of the spinal cord hindlimb enlargement in low-spinal turtles. Forward swimming was evoked in the left hindlimb by electrical stimulation of the right dorsolateral funiculus (DLF) at the anterior end of the third postcervical spinal segment (D3). Animals were held by a band-clamp in a water-filled tank so that hindlimb movements could be recorded from below with a digital video camera. Left hindlimb hip and knee movements were tracked while electromyograms (EMGs) were recorded from left hip and knee muscles. In turtles with intact spinal cords, electrical stimulation of the right D3 DLF evoked robust forward swimming movements of the left hindlimb, characterized by rhythmic alternation between hip flexor (HF) and hip extensor (HE) EMG discharge, with knee extensor (KE) bursts occurring during the latter part of each HE-off phase. After removing the right spinal hemi-enlargement (D8-S2), DLF stimulation still evoked rhythmic locomotor movements and EMG bursts in the left hindlimb that included HF-HE alternation and KE discharge. However, post-surgical movements and EMG bursts had longer cycle periods, and movements showed lower amplitudes compared to controls. These results show that (1) sufficient locomotor CPG circuitry resides within the turtle spinal hemi-enlargement to drive major components of the forward swim motor pattern, (2) contralateral circuitry contributes to the excitation of the locomotor CPG for a given limb, and (3) a sufficient portion of the descending DLF pathway crosses over to the contralateral cord anterior to the hindlimb enlargement to activate swimming.

In vivo prompt gamma neutron activation analysis for the screening of boron-10 distribution in a rabbit knee: a simulation study.

Boron neutron capture synovectomy (BNCS) is under development as a potential treatment modality for rheumatoid arthritis (RA). RA is characterized by the inflammation of the synovium (the membrane lining articular joints), which leads to pain and a restricted range of motion. BNCS is a two-part procedure involving the injection of a boronated compound directly into the diseased joint followed by irradiation with a low-energy neutron beam. The neutron capture reactions taking place in the synovium deliver a local, high-linear energy transfer (LET) dose aimed at destroying the inflamed synovial membrane. For successful treatment via BNCS, a boron-labeled compound exhibiting both high synovial uptake and long retention time is necessary. Currently, the in vivo uptake behavior of potentially useful boronated compounds is evaluated in the knee joints of rabbits in which arthritis has been induced. This strategy involves the sacrifice and dissection of a large number of animals. An in vivo (10)B screening approach is therefore under investigation with the goal of significantly reducing the number of animals needed for compound evaluation via dissection studies. The 'in vivo prompt gamma neutron activation analysis' (IVPGNAA) approach uses a narrow neutron beam to irradiate the knee from several angular positions following the intra-articular injection of a boronated compound whose uptake characteristics are unknown. A high-purity germanium detector collects the 478 keV gamma photons produced by the (10)B capture reactions. The (10)B distribution in the knee is then reconstructed by solving a system of simultaneous equations using a weighted least squares algorithm. To study the practical feasibility of IVPGNAA, simulation data were generated with the Monte Carlo N-particle transport code. The boron-containing region of a rabbit knee was partitioned into 8 compartments, and the (10)B prompt gamma signals were tallied from 16 angular positions. Results demonstrate that for this level of spatial resolution, an estimate of (10)B distribution inside the joint can be obtained to within 10% uncertainty, under ideal conditions. Variations of the anatomic dimensions among individual rabbit knees and potential knee positioning errors will result in an uncertainty of over 20%. IVPGNAA thus provides sufficient resolution and quantification regarding the in vivo uptake characteristics of boronated pharmaceuticals to serve as a useful means of screening new compounds of potential use in BNCS.

Dirhenium decacarbonyl-loaded PLLA nanoparticles: influence of neutron irradiation and preliminary in vivo administration by the TMT technique.

In a previous study, we have described the elaboration of PLLA-based nanoparticles loaded with non radioactive dirhenium decacarbonyl [Re(2)(CO)(10)], a novel neutron-activatable radiopharmaceutical dosage form for intra-tumoral radiotherapy. These nanoparticles are designed for a neutron irradiation which can be carried out in a nuclear reactor facility. This new paper describes the neutron irradiation influence on these Re(2)(CO)(10)-loaded PLLA nanoparticles. The loaded nanoparticles with 23% (w/w) of metallic rhenium have shown to remain stable and separated and to keep out their sphericity at the lower neutron flux (1x10(11)n/cm(2)/s for 0.5h) which was used for rhenium content determination (neutron activation analysis, NAA). However, when loaded nanoparticles were irradiated at the higher neutron flux (1.45x10(13)n/cm(2)/s, 1h), they have shown to be partially coagglomerated and some pores appeared at their surface. Furthermore, DSC results showed a decrease in the PLLA melting point and melting enthalpy in both blank and loaded nanoparticles indicating a decrease in polymer crystallinity. In addition, the polymer molecular weights (M(n), M(w)) decreased after irradiation but without largely affecting the polymer polydispersity index (P.I.) which indicated that an irradiation-induced PLLA chain scission had occurred in a random way. The XRD patterns of irradiated PLLA provided another proof of polymer loss of crystallinity. FTIR spectra results have shown that irradiated nanoparticles retained the chemical identity of the used Re(2)(CO)(10) and PLLA despite the reduction in polymer crystallinity and molecular weight. Nanoparticles suspending after irradiation became also more difficult, but it was properly achievable by adding PVA (1%) and ethanol (10%) into the dispersing medium. Moreover, after 24h incubation of different irradiated nanoparticles in two different culture mediums, visual examination did not show bacterial growth indicating that applied neutron irradiation, yielding an absorbed dose of 450kGy, can be a terminal method for nanoparticles sterilisation. Thereafter, in a preliminary in vivo experiment, superparamagnetic non radioactive nanoparticles loaded with Re(2)(CO)(10) and oleic-acid coated magnetite have been successfully injected into a mice animal model via targeted multi therapy (TMT) technique which would be our selected administration method for future in vivo studies. In conclusion, although some induced neutron irradiation damage to nanoparticles occurs, dirhenium decacarbonyl-loaded PLLA nanoparticles retain their chemical identity and remain almost as re-dispersible and injectable nanoparticles by the TMT technique. These nanoparticles represent a novel interesting candidate for local intra-tumoral radiotherapy.

Effect of chronic intermittent exposure to AM radiofrequency field on responses to various types of noxious stimuli in growing rats.

There are several reports of altered pain sensation after exposure (from a few minutes to hours in single or repeated doses for 2-3 weeks) to electromagnetic fields (EMF) in adults. The commonly utilized noxious stimulus is radiant heat. The nociceptive responses are known to be influenced by characteristics of stimulus, organism, and environment. We studied the pattern of nociceptive responses to various noxious stimuli in growing rats exposed to radiofrequency field (73.5 MHz amplitude modulated, 16 Hz power density 1.33 mw/cm(2), SAR = 0.4 w/kg) for 45 d (2 h/d). Threshold current for stimulation of nociceptive afferents to mediate motor response of tail (TF), vocalization during stimulus (VD), and vocalization after discharge (VA); the withdrawal latency of tail (TFL) and hind paw (HPL) to thermal noxious stimulus and tonic pain responses were recorded in every rat. The TFL was not affected, HPL was decreased (p < 0.01), and the thresholds of TF and VD were not affected, while, that of VA was significantly decreased. The tonic pain rating was decreased (p < 0.01). A decrease in the threshold of VA (p < 0.01) is indicative of an increase in the emotional component of the response to the phasic pain, whereas a decrease in the pain rating indicates analgesia in response to the tonic pain. The results of our study suggest that chronic (45 d), intermittent (2 h/d) amplitude modulated RF field exposure to the peripubertal rat increases the emotional component of phasic pain over a basal eaualgesic state, while late response to tonic pain is decreased. The data suggest that amplitude modulated RF field differentially affects the mechanisms involved in the processing of various noxious stimuli.