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Background and Objectives: With the increasing use of fluorescence in medical applications, a comprehensive understanding of the effect of temperature on tissue autofluorescence is essential. The purpose of this study is to explore the effect of temperature on the fluorescence of porcine cornea and rat skin and determine the relative contributions of irreversible changes in optical properties and in fluorescence yield., Study Design/materials and Methods: Fluorescence, diffuse reflectance, and temperature measurements were acquired from excised porcine cornea and rat skin over a temperature range of 0-80 °C. A dual excitation system was used with a 337 nm pulsed nitrogen laser for the fluorescence and a white light source for the diffuse reflectance measurements. A thermal camera measured tissue temperature. Optical property changes were inferred from diffuse reflectance measurements. The reversibility of the change in fluorescence was examined by acquiring measurements while the tissue sample cooled from the highest induced temperature to room temperature., Results: The fluorescence intensity decreased with increasing tissue temperature. This fluorescence change was reversible when the tissue was heated to a temperature of 45 °C, but irreversible when heated to a temperature of 80 °C., Conclusion: Auto-fluorescence intensity dependence on temperature appears to be a combination of temperature-induced optical property changes and reduced fluorescence quantum yield due to changes in collagen structure. Temperature-induced changes in measured fluorescence must be taken into consideration in applications where fluorescence is used to diagnose disease or guide therapy., (Copyright © 2012 Wiley Periodicals, Inc.)

Biodegradable drug-delivery systems can be formulated to release drug for hours to years and have been used for the controlled release of medications in animals and humans. An important consideration in developing a drug-delivery matrix is knowledge of the long-term stability of the form of the drug and matrix after formulation and any changes that might occur to the drug throughout the delivery process. Solid-state NMR spectroscopy is an effective technique for studying the state of both the drug and the matrix. Two systems that have been studied using solid-state NMR spectroscopy are presented. The first system studied involved bupivacaine, a local anesthetic compound, which was incorporated into microspheres composed of tristearin and encapsulated using a solid protein matrix. Solid-state 13C NMR spectroscopy was used to investigate the solid forms of bupivacaine in their bulk form or as incorporated into the tristearin/protein matrix. Bupivacaine free base and bupivacaine-HCl have very different solid-state NMR spectra, indicating that the molecules of these compounds pack in different crystal forms. In the tristearin matrix, the drug form could be determined at levels as low as 1:100 (w/w), and the form of bupivacaine was identified upon loading into the tristearin/protein matrix. In the second case, the possibility of using solid-state 13C NMR spectroscopy to characterize biomolecules lyophilized within polymer matrices is evaluated by studying uniformly 13C-labeled asparagine (Asn) in 1:250 (w/w) formulations with poly(vinyl pyrrolidone) (PVP) and poly(vinyl alcohol) (PVA). This work shows the capability of solid-state NMR spectroscopy to study interactions between the amino acid and the polymer matrix for synthetic peptides and peptidomimetics containing selective 13C labeling at the Asn residue.

Purpose: This investigation determines the drug delivery capacity of Lipospheres, which are drug-containing solid-filled vesicles made of triglyceride with a phospholipid outer covering, to release local anesthetic in vitro and to produce sustained peripheral nerve block in vivo., Methods: The local anesthetic, bupivacaine, was loaded into Lipospheres in several dosage forms, characterized, and measured for in vitro release. In rats, Lipospheres were administered into a large space between muscle layers surrounding the sciatic nerve to assess sensory and motor block in vivo., Results: The particle size of Lipospheres was determined to be between 5 and 15 microm, with over 90% surface phospholopid. Lipospheres released bupivacaine over two days under ideal sink conditions. Liposphere nerve application produced dose-dependent and reversible block. Indeed, sustained local anesthetic block (SLAB) was observed for 1-3 days in various in vivo tests: a) Hind paw withdrawal latency to noxious heat was increased over 50% for 96 hr period after application of 3.6% or 5.6% bupivacaine-Lipospheres. The 3.6% and 5.6% doses were estimated to release bupivacaine at 200 and 311 microg drug/ hr, respectively, based on release spanning 72 hr. Application of 1.6% bupivacaine-Lipospheres increased withdraw latency 25-250% but for only a 24 hr duration; b) Similarly, vocalization threshold to hind paw stimulation was increased 25-50% for 72 hr following application of 3.6% bupivacaine-Lipospheres; c) Finally, sensory blockade outlasted or equaled corresponding motor block duration for all Liposphere drug dosages., Conclusions: Liposphere delivery of local anesthetic drugs may be well suited for site-specific pharmacotherapy of neural tissue to produce SLAB. Dose-dependent effects in duration of action may include lipophilic tissue storage.

Increased GABA activity in the medial hypothalamus (HYP) and midbrain central gray (MCG), but not the preoptic area (POA), facilitates sexual receptivity in the female rat [40]. In the current experiments, ovariectomized females were chronically treated with estrogen (via silastic capsules) to maintain a continuously high level of lordosis response. Administration of crystalline antisense oligodeoxynucleotide to the GABA synthetic enzyme, GAD67, into the HYP and MCG significantly and reversibly reduced lordosis response for 1-2 days, but did not inhibit lordosis when administered into the POA. Administration of a control oligonucleotide, consisting of the same nucleotide bases but in a scrambled sequence, did not significantly modulate behavior when infused into any brain areas. When oligodeoxynucleotide antisense to GAD67 was suspended in oil and then infused into the HYP or MCG it was more effective and resulted in less inter-animal variability. Subsequent experiments involving infusions into the MCG compared the effectiveness of antisense oligonucleotides to the two different forms of GAD, known as GAD65 and GAD67. Oligodeoxynucleotides antisense to the mRNA for either gene were effective at reducing lordosis behavior but with a different time course. Oligonucleotide antisense to GAD67 significantly reduced behavior within 24 h of infusion and there was full recovery by 4 days post-infusion. GAD65 antisense oligonucleotide did not significantly reduce behavior until 48 h post infusion and animals did not fully recover to pretest levels of lordosis until 5 days post-infusion. When antisense oligonucleotide for the two genes was administered simultaneously, the inhibition of lordosis was maximal at 24 h and stayed depressed for 4 days. There did not appear to be an additive effect of the two different antisense oligonucleotides when administered together. Tissue GABA levels in HYP and MCG of individual rats assayed by HPLC were no longer correlated with lordosis score after antisense oligonucleotide infusion but were after infusions of scrambled control oligos. Immunoblotting for the two forms of GAD revealed that GAD67 antisense oligonucleotide infusion led to significant decreases in both GAD67 and GAD65 protein levels as compared to infusions of scrambled control oligo. In addition, the levels of a neuronal marker, neuron-specific enolase, also decreased (although nonsignificantly) suggesting either a temporary shutdown of protein synthesis or a degeneration of GABAergic neurons after GAD67 antisense oligonucleotide infusion.

Polyanhydride polymer matrices have been used successfully for sustained release of a number of drugs in vitro and in vivo. Dibucaine free base, dibucaine HCl, and bupivacaine HCl were incorporated into polymer matrices with copolymer 1,3-bis(p-carboxyphenoxy)propane-sebacic acid anhydride (1:4). Drug release was measured in vitro following incubation of the drug-polymer matrices in phosphate buffered solution, pH 7.4, at 37 degrees C, to approximate in vivo conditions. Local anesthetics were released in a sustained manner yielding 90% cumulative drug release over periods ranging from 3 to 14 days. The kinetics of release varied with both the choice of local anesthetic and the method of drug incorporation into the matrix (hot melt versus compression molding). Polymer local anesthetic matrix devices (PLAM), loaded by hot melt incorporation with 20% bupivacaine, were implanted in vivo adjacent to the sciatic nerve in three rats. Reversible neural blockade was observed for 4 days in all animals. Polymer implants without local anesthetic showed no neural blockade. This technology could lead to methods of prolonged blockade of peripheral nerves or of sympathetic ganglia, which may be utilized for the management of postoperative pain, sympathetically maintained pain, or certain forms of chronic pain.

Based on pharmacological evidence that inhibitory amino acids mediate vaginocervical mechano-stimulation produced analgesia (VSPA), we hypothesized that inhibitory amino acids would be released endogenously in the spinal cord in response to vaginocervical mechano-stimulation (VS). This hypothesis was tested by HPLC analysis of the amino acid content of 5-min superfusates of the spinal cord before, during and after VS (400 g force applied against the cervix) in urethane-anesthetized rats. Utilizing an in vivo push-pull superfusion method, artificial cerebrospinal fluid was continuously superfused over the spinal cord through the intrathecal space surrounding the sacral-lower thoracic region. In addition, concentrations of amino acids in the superfusate were measured in response to KCl stimulation (increasing the superfusion medium from 3.4 to 40.0 mM KCl to produce non-specific depolarization), and noxious hind paw mechano-stimulation (pinching the hind paw to produce a sustained flexor response in ipsilateral hind leg). There was a significant increase in the concentration of Gly, Tau, Asp, Glu and Lys in the superfusate in response to VS (n = 8) and to KCl (n = 8), but not to hind paw stimulation (n = 5). Also, GABA concentrations increased in response to KCl, and the concentration of Ala, Ser, Gln, Thr, Arg and Phe increased in response to VS, however, GABA levels were sometimes below the limits of detection. In contrast, there was no significant change in any amino acid concentration in response to hind paw pinch stimulation, and VS did not significantly affect the concentrations of Tyr, His, Ile, Leu, Met, Trp or Val. The present findings support our hypothesis that VS releases inhibitory amino acids in the spinal cord. Moreover, other amino acids, including 'excitatory' amino acids, are released into the superfusate. The profile of amino acid release in response to VS differs from that in response to paw pinch or KCl administration.

Background: Prolonged nerve blockade is potentially useful in the management of many acute and chronic pain problems. Aside from infusions via an indwelling catheter, most currently available nondestructive techniques for prolonging local anesthetic action cannot provide more than 1-2 days of blockade. Bioerodible polymer matrixes have been used to deliver a variety of drugs in patients and animals for periods lasting weeks to years. Previously, dibucaine and bupivacaine were incorporated into copolymers of 1,3 bis(p-carboxyphenoxy) propane-sebacic acid anhydride (1:4), and demonstrated sustained release in vitro following incubation of the drug-polymer matrixes in phosphate-buffered solution (pH 7.4, 37 degrees C)., Methods: In the present study, cylindrical pellets made from polymer matrixes incorporated with bupivacaine-HCl were implanted surgically along the sciatic nerves of rats. Neural block was assessed by direct observation of motor skills and by leg-withdrawal latency to a hot surface. Biochemical and histologic examinations were performed 2 weeks after implantation., Results: Sensory and motor blockade was produced for periods ranging from 2 to 6 days. Contralateral control legs receiving polymer implants without drug showed no block. Blockade was reversible, and animals appeared to recover sensory and motor function normally. Biochemical indexes of nerve and muscle function were indistinguishable from contralateral controls., Conclusions: This biodegradable polymer system provides a promising new alternative for the delivery of local anesthetics to peripheral nerves to produce prolonged blockade for the management of acute and chronic pain.

To increase sensitivity and to improve normalization of RNA levels in Northern blot analysis, a comparatively inexpensive optical scanner was utilized for digitizing photonegatives of ethidium bromide stained gels and autoradiograms. The optical scanner captures the image with a maximum resolution of 300 dots per inch by assigning one of 256 gray levels (8-bit) to each dot in the image. With the use of the public domain NIH Image program (requires a Macintosh II and an 8-bit video card), gel or autoradiogram bands in the digitized image are selected and their average gray scale density measured. We found that the digitized image of a photonegative of a TAE (Tris-acetate/EDTA) agarose gel, loaded incrementally with 50-1500 ng total RNA, produced a linear response over a 4-fold range down to 100 ng (R2 greater than 0.950). In utilizing "quantification" gels like this, RNA samples that are too dilute or too small for traditional spectrophotometric techniques can be normalized and loaded uniformly onto subsequent Northern gels. Results from autoradiogram scans demonstrate highly linear gray scale responses over a 4-fold range of total RNA (R2 greater than 0.950) that are reproducible with different blots and probe types (e.g., riboprobe, cDNA and oligonucleotide). In addition, we describe a normalization technique using a 30-mer oligonucleotide probe for rat 28S ribosomal RNA as a measure of total RNA loaded per gel lane. Altogether, this scanning, ribosomal RNA normalization system allows the measurement of relative changes between 20% and 400% using standard autoradiographic methods.

Neonatal capsaicin treatment (50 mg/kg SC in two-day-old rats) increased thermal pain thresholds in both sexes when measured at different ages, decreased the responsiveness of adult females to specific noxious stimulation, and differentially decreased the magnitude of vaginocervical stimulation (VS)-produced analgesia in nociceptive tests. When adult, "capsaicin" females (n = 37) were significantly greater than controls (n = 24) in vocalization threshold (VT) to electrical tail shock (55.4%) and in paw lick (PL) latency to a hot plate (75.9%). In contrast, neither tail flick (TF) latency nor the leg withdrawal reflex (LWR) to mechanical pressure of the ipsilateral hind paw was affected by neonatal capsaicin. In response to VS, the controls showed a significant increase in thermal (TF, 279%; PL, 411%), mechanical pressure (LWR, 100%) and electrical (VT, 86.8%) pain thresholds. The "capsaicin" females response to VS was significantly less than controls in TF (26.1%), PL (26.0%), and LWR (54.1%) measures, and surprisingly, during VS their VT was significantly decreased below baseline levels 12.2% +/- 4.3. These results suggest that neonatal capsaicin treatment differentially attenuates the analgesia-producing component of VS, while sparing a nociception-inducing component of this stimulus. That is, after neonatal capsaicin treatment, the ability of VS to produce analgesia is reduced; moreover, VS lowers the VT, suggesting that it actually becomes a noxious stimulus in and of itself.

GABAergic neurotransmission has been implicated in the control of the steroid-dependent behavior, lordosis. GABA has dual effects on lordosis: it facilitates lordosis through actions in the medial hypothalamus (mHYP) and it inhibits lordosis through actions in the preoptic area (POA). In the present study, gonadally intact and ovariectomized female rats were behaviorally tested with a sexually active male. Brains were removed from sexually receptive female either 1 or 24 h after behavioral testing. There was a significant difference in endogenous GABA concentration in HYP and POA between receptive, postreceptive and ovariectomized nonreceptive females. Specifically, GABA levels in postreceptive females were higher in the HYP (20%) and lower in the POA (21%) in comparison to receptive females (p less than 0.05). There was also a significant change in binding parameters of 3H-muscimol in the HYP and POA of receptive females as compared to 24 h postreceptive and ovariectomized rats. Attempts to modulate 3H-GABA release from hypothalamic tissue slices by estrogen or progesterone in ovariectomized rats yielded no effect on this parameter.

Phencyclidine (PCP), haloperidol, and naloxone were administered alone and in combination to rats responding under a fixed-interval schedule for water presentation. Lower doses of PCP (0.25-2.0 mg/kg) and naloxone (0.001-0.1 mg/kg) produced increases while higher doses produced dose-dependent decreases in response rate. Haloperidol (0.0625-0.5 mg/kg) produced a monotonic dose-dependent decrease in responding. When a dose of naloxone (8.0 mg/kg) that did not alter responding was administered prior to the PCP, the PCP dose-response curve was shifted to 6.5-fold lower doses of PCP. When a dose of haloperidol (0.0625 mg/kg) that did not alter responding was administered prior to the PCP, the PCP dose-response curve was shifted to 1.5-fold higher dose of PCP. These observations are discussed in relation to current views of the mechanism of PCP action.

The tolerance which develops following the repeated administration of phencyclidine has been attributed to both pharmacological and behavioral adaptations. In the present study, the possibility that neurochemical alterations induced by the repeated administration of phencyclidine might account for some portion of the observed tolerance was examined. Specifically, it was postulated that the repeated administration of high doses of phencyclidine might result in long lasting depletion of central catecholamine stores and that this neurochemical perturbation might result in tolerance to the drug's effects. It was observed that phencyclidine disrupted the performance of rats on a fixed-interval schedule maintained by water presentation. The repeated administration of high doses of phencyclidine (but not of a comparable volume of saline) during a period when subjects were not engaged in the fixed-interval task resulted in the development of a long lasting tolerance, but this tolerance could not be accounted for in terms of a depletion of central catecholamine stores.

Ovariectomized adult C57BL/6J mice were exposed to androgens, estrogens, or combined androgen-estrogen treatments and tested for the display of male-typical aggressive behavior toward olfactory bulbectomized stimulus males. Among the androgenic treatments (testosterone, dihydrotestosterone, or methyltrienolone) only testosterone, which, in contrast to the other androgens, can be aromatized, activated fighting behavior. In the second experiment, estradiol benzoate (EB) was totally ineffective as an aggression-promoting compound. Lastly, combined EB+dihydrotestosterone also did not induce male-like aggression. These data suggest that T itself may be capable of promoting aggression without undergoing aromatization or 5 alpha-reduction.

In vivo push-pull superfusion was used to sample the regional release of amino acids into spinal superfusates of urethane-anesthetized rats. By collecting superfusates from the intrathecal space surrounding the sacral-lower thoracic spinal cord, it was possible to achieve a stable release of amino acids in one- and five-minute superfusate fractions. Introducing the depolarizing agent, potassium chloride (KC1) (40 mM), into the superfusion medium significantly increased GLU, GLY, and TAU concentrations in superfusates compared to pre-KCl values. The findings that these three amino acids were the only ones (out of 20) that showed a significant increase in response to KCl administration, suggest that they mediate neurotransmission in this region of the spinal cord. Amino acid concentrations were determined in spinal superfusates by high performance liquid chromatography (HPLC), utilizing an automated ortho-phthaldialdehyde precolumn derivatization system. The regional superfusion system described in this paper provides a technique for measuring KC1-produced release of neurochemicals that may mediate neurotransmission in delimited spinal regions.